SINGLET OXYGEN FORMATION BY SENSITIZATION OF FUROCOUMARINS COMPLEXED WITH, OR BOUND COVALENTLY TO DNA

Abstract— The formation of singlet molecular oxygen (¹O₂) by sensitization of the furocoumarins 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen complexed with DNA was investigated. From the results it is concluded that 5-MOP complexed with native DNA is able to generate ¹O₂, even in a larger extent than 5-MOP free in solution. Also, with 8-MOP and especially with psoralen, ¹O₂ formation by the complexed compound could be observed. The ¹O₂ formation sensitized by covalently bound furocoumarin was demonstrated with psoralen as a model compound. 4',5'-Dihydropsoralen, a model compound for the UVA light absorbing 4',5'monoadducts of furocoumarins to DNA, is also able to generate ¹O₂.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.     

EFFECT OF SODIUM AZIDE ON PHOTODYNAMIC INDUCTION OF GENETIC CHANGES IN YEAST

Abstract— In view of the increasing attention to ¹O₂ (¹Δ_g) participation in the photodynamic action, different types of genetic changes in Saccharomyces cerevisiae by acridine orange sensitization were compared with respect to the response to N₃^-, a well known quencher of ¹O₂. The induction of mitotic crossing over with respect to ade 2 locus and mitotic gene conversion at trp 5 locus were suppressed by the addition of N₃^- suggesting the involvement of ¹O₂ as a major intermediate. However, the induction of reverse mutation at ilv 1 was only slightly suppressed. These results may indicate that there are two types of photodynamic DNA damage; one is produced via ¹O₂ and the other via non-¹O₂ reaction pathway which lead to mitotic gene conversion and mitotic crossing over, and to mutation, respectively.     

UVA self-photosensitized oxygenation of beta-ionone

The steady-state UVA (350 nm) photolysis of ( E )-β-ionone ( 1 ) in aerated toluene solutions was studied by ¹H NMR spectroscopy. The formation of the 1,2,4-trioxane ( 2 ) and 5,8-endoperoxide ( 5 ) derivatives in the ratio of 4:1 was observed. Time-resolved laser induced experiments at 355 nm, such as laser-flash photolysis, photoacoustic and singlet oxygen ¹O₂ phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield Φ _T = 0.50 as the precursor for the generation of singlet oxygen ¹O₂ ( Φ _Δ = 0.16) and the isomeric α-pyran derivative ( 3 ), which was a reaction intermediate detected by NMR. In turn, the reaction of ¹O₂ with 1 and 3 occurred with rate constants of 1.0 × 10⁶ and 2.5 × 10⁸  m ⁻¹s⁻¹ to yield the oxygenated products 5 and 2 , respectively, indicating the relevance of the fixed s-cis configuration in the α-pyran ring in the concerted [2+4] cycloaddition of ¹O₂.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

A NEW METHOD FOR THE DETECTION OF SINGLET OXYGEN IN AQUEOUS SOLUTIONS

Abstract— In experiments on the interception of reactive intermediates of strongly oxidizing character in dye (S) sensitized photooxidations using p -nitrosodimethylaniline (RNO) as a selective scavenger, it has been observed that some substrates (A) or ¹O₂ acceptors (like imidazole derivatives) induce the bleaching of RNO as followed spectrophotometrically at 440 nm. Since singlet oxygen (¹O₂) does not react chemically with RNO, this bleaching is a consequence of ¹O₂ capture by the imidazole ring which results in the formation of a trans-annular peroxide intermediate [¹O₂] capable of inducing the bleaching of RNO (-RNO). In the absence of RNO, [¹O₂] decomposes or rearranges into the final oxygenation product ¹O₂: ¹Δ_g Thus, the system imidazole plus RNO can be used as a sensitive and selective test for the presence of ¹O₂ in aqueous solutions. The method can also be applied in the presence of sensitizing dyes which, under visible irradiation, can partially bleach RNO even in the absence of imidazole derivatives. In such a case, the bleaching of RNO is strongly increased by the presence of imidazoles with a characteristic dependence on their concentration. The separation of the product of RNO bleaching by thin layer chromatography can serve as additional proof of the presence of ¹O₂ in the system. The imidazole plus RNO method has been applied to a number of sensitizing and non-sensitizing dyes.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

EVIDENCE FOR THE PRODUCTION OF SINGLET OXYGEN BY PHOTOEXCITED ACRIDINE ORANGE

Abstract In contrast to previous results obtained with the nitroxide radical detection technique, generation of the specific ¹O₂ oxidation product of cholesterol shows that photoexcited acridine orange produces singlet oxygen.     

MECHANISMS FOR DYE-MEDIATED PHOTODYNAMIC ACTION: SINGLET OXYGEN PRODUCTION, DEOXYGUANOSINE OXIDATION AND PHAGE INACTIVATING EFFICIENCIES

Abstract The production of singlet oxygen (¹O₂) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6-tetramethyl-4-piperidone as ¹O₂ acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of ¹O₂ production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of ¹O₂ was not observed with 9-aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the ¹O₂ production and (ii) the bacteriophage ØX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated ¹O₂. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.     

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

RELATION BETWEEN SOME PHOTOBIOLOGICAL PROPERTIES OF FUROCOUMARINS AND THEIR EXTENT OF SINGLET OXYGEN PRODUCTION

Abstract— The production of singlet oxygen (¹O₂) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8-methoxypsoralen (8-MOP) to generate ¹O₂.
The following compounds: 5-methoxypsoralen (5-MOP), psoralen, 4,5',8-trimethylpsoralen (TMP) and 5,8–dimethoxypsoralen (5,8–DMOP), are able to generate ¹O₂ when irradiated with long–wave ultraviolet light. With the photobiologically inactive angelicin no ¹O₂ production has been found. The relative extent of ¹O₂ formation has been determined for the various furocoumarins and has been compared with literature data for the skin photosensitizing effect. The observed relation between experimental data on the one side and the literature data on the other side is discussed.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.     

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (¹O₂) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of ¹O₂ from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in ¹O₂ generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0₂ quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by ¹O₂ which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of ¹O₂ was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of ¹O₂.     

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (¹O₂) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub-mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe-S centers of nonheme iron proteins. Disruption of the Fe-S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of ¹O₂ generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe-S centers to the generation of ¹O₂ from SMP, we suggest that the Fe-S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of ¹O₂ is also discussed.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.     

A PRELIMINARY INVESTIGATION OF THE USE OF LIMONENE FOR DETECTING SINGLET OXYGEN AS A COMPONENT OF POLLUTED AIR

Abstract— Exploratory experiments using simulated conditions indicate that the terpene, (+)-limonene can serve to detect O₂(¹Δ_g) in polluted atmospheres. ¹O₂ attack on limonene results in the formation of specific oxidation products in a specific distribution; quantitative identification of these products is a highly specific test for ¹O₂.