THE ROLE OF SINGLET OXYGEN IN PHOTOOXIDATION OF EXCITABLE CELL MEMBRANES

Abstract— Giant nerve axons from lobsters were photooxidized by visible light from a Xe arc using eosin Y, rose bengal and methylene blue as sensitizers. The degree of modification was assayed as the rate constant for block of sodium channels during illumination. Protection or enhancement of modification was tested for several reagents known to interact with singlet oxygen. Deuterium oxide enhanced the modification up to 57% while sodium azide protected up to 60%. β -Carotene, triethylamine and DABCO exerted no effect. From a consideration of the probable low polarity environment of the sensitization sites and diffusional restrictions on reagents it is concluded that intramembranously created singlet oxygen plays a significant role in membrane modification.     

A TEST OF THE SINGLET OXYGEN MECHANISM OF CATIONIC DYE PHOTOSENSITIZATION OF MITOCHONDRIAL DAMAGE

Aromatic cationic dyes have a potential as photo-chemotherapeutic agents because they are selectively concentrated into the mitochondria of cancerous cells. The mechanism of cytophototoxicity has been proposed to be primarily due to dye sensitized photogeneration of highly toxic singlet oxygen (1O2) at the mitochondria. We tested this hypothesis by measuring the relative phototoxicity of a collection of aromatic cationic dyes towards respiring rat-liver mitochondria (RLM), upon addition of 514 nm laser light. Effectiveness of dye photosensitization towards destruction of RLM function was assayed by its effect on the RLM membrane potential. Three physical parameters of dye phototoxicity were independently measured and a relative phototoxicity calculated assuming adherence of mechanism to the 1O2 hypothesis. Quantum yields of dye sensitized 1O2 production were estimated, either from time-resolved luminescence measurements of photosensitized 1O2 formed, or by comparing rates of photobleaching of 1O2 trap; the relative partition of dye into mitochondrial lipid was determined gravimetrically; and the optical density of dye was determined in a lipid like Triton X-100 micellar environment. Under the assumption of the 1O2 hypothesis, these parameters were used to predict a relative phototoxicity which was compared with that observed. For 12 of the 14 dyes investigated, the observed and predicted phototoxicities were linearly correlated (r = 0.85) suggesting support of the 1O2 hypothesis. Carbocyanines DiOC2(3) and DiSC2(3) did not correlate and were found to be 10 and 1000 times more potent than predicted, suggesting an additional factor at play in their phototoxicity.     

OXIDATION OF PHOSPHATIDYLCHOLINE MEMBRANES BY SINGLET OXYGEN GENERATED IN THE GAS PHASE

Singlet-oxygen (1O2) was generated in the gas phase by heterogeneous photosensitization and bubbled into suspensions of phosphatidylcholine (PC) liposomes. Lipid peroxidation and membrane lysis were observed, and were dependent on the 1O2 concentration and the degree of unsaturation of the liposome. An analysis based on large target diffusion theory indicates that approximately 5000, 2800, and 1600 interactions were required for the lysis of large dioleoylPC, dilinoleoylPC and dilinolenoylPC liposomes, respectively.     

PROTECTION BY DABCO AGAINST INACTIVATION OF TRANSFORMING DNA BY NEAR-ULTRAVIOLET LIGHT: ACTION SPECTRA AND IMPLICATIONS FOR INVOLVEMENT OF SINGLET OXYGEN

Abstract— Diazobicyclo (2.2.2) octane (DABCO) protects the genetic activity of purified transforming Bacillus subtilis DNA against inactivation by near-, but not far-, UV light. The maximum dose-modifying factor is 0.4, at 0.1 M DABCO. Maximal protection is at about 350 nm and no protection occurs below 313 nm. The spectrum for protection is similar to that described for 2-aminoethylisothiouronium bromide hydrobromide. The relevance of these observations with regard to the role of singlet oxygen in near-UV effects is discussed.     

几种血卟啉3,8-烷氧(硫)基衍生物产生单重态氧能力的研究

在“血卟啉衍生物”(HPD)^[1]肿瘤光动力疗法取得令人瞩目成就的同时,化学家们证明了临床使用的HPD制剂并非原意义上的血卟啉衍生物,而是一种复杂的卟啉混合物^[2],其中血卟啉(HP),羟乙基-乙烯基-次卟啉(HVD),原卟啉(PP)约占总量的80%,Dougherty等分离得到其余20%未知结构羧基卟啉,认为此为HPD的有效成分,经进一步分析测定其主要成分是二血卟啉醚(DHE)^[3]。     

CUTANEOUS PHOTOTOXICITY OF TETRACYCLINE ANTIBIOTICS: GENERATION OF FREE RADICALS and SINGLET OXYGEN DURING PHOTOLYSIS AS MEASURED BY SPIN-TRAPPING and THE PHOSPHORESCENCE OF SINGLET MOLECULAR OXYGEN

Chlortetracycline (CTC) generated an aryl radical in aqueous buffer (pH 7.4) during near UV irradiation, as evidenced by the formation of 2-methyl-2-nitrosopropane spin adducts. The radical was produced via dechlorination, a photoprocess not previously reported for tetracyclines. Demeclocyc-line (DEM), another chlorinated tetracycline, did not produce detectable aryl radicals. Relative ¹O₂ yields obtained by direct luminescence measurements at 1268 nm for five tetracyclines in alkaline ethanol (demeclocycline · tetracycline · chlortetracycline · doxycycline · minocycline) showed that DEM produced approximately three times as much singlet oxygen as CTC. This constitutes direct evidence that tetracyclines sensitize both Type I and Type II photoreactions.     

SINGLET OXYGEN GENERATION BY PORPHYRINS AND THE KINETICS OF 9,10-DIMETHYLANTHRACENE PHOTOSENSITIZATION IN LIPOSOMES

Abstract— Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn²⁺- and MG²⁺-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (Φ_Δ) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn²⁺-phthalocyanine tetrahydroxyl [ZnPC(OH)₄] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the Φ_Δ values were evaluated. At 10 μ M sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of Φ_Δ (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)₄ (0.005). As a test of the method, Φ_Δ for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)₄ (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.     

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.     

THE PHOTOCHEMICAL YIELD OF SINGLET OXYGEN FROM PORPHYRINS IN DIFFERENT STATES OF AGGREGATION

Abstract— Aqueous solutions of hematoporphyrin and hematoporphyrin derivatives were exposed to light. When present in such solutions tryptophan is degraded by a singlet oxygen mechanism. This is true for excitation at 396 nm, where porphyrin monomers have their absorption maximum, as well as for excitation at 360 nm, where porphyrin aggregates seem to absorb strongly. The quantum yield of singlet oxygen production is similar within 25% for excitation at 396 and 360 nm while the fluorescence quantum yield is more than a factor 2 lower for excitation at 360 nm than for excitation at 396 nm. Photoexcitation of the clinically used hematopotophyrin derivatives photofrin I and photofrin II produces singlet oxygen with significantly smaller yields than photoexcitation of hematoporphyrin. Thus, the aggregates present in solutions of photofrin I and photofrin II are of a different nature than those present in aqueous solutions of hematoporphyrin.     

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.     

INVOLVEMENT OF SINGLET OXYGEN IN THE PHOTOTOXICITY MECHANISM FOR A METABOLITE OF PIROXICAM

It has been previously shown that a metabolite of piroxicam but not piroxicam itself causes phototoxicity to cells in vitro after exposure to UVA (320–400 nm) radiation. The phototoxicity mechanism for this metabolite, 2-methyl-4-oxo-2H-l,2-benzothiazine-l,l-dioxide (Compound I), was investigated. In vitro phototoxicity to human mononuclear cells was assayed using 0.5 m M Compound I and UVA radiation. The UVA fluence required for phototoxicity of Compound I was lower by a factor of 2-3 in D₂O buffer compared to H₂O buffer. Superoxide dismutase and mannitol, which remove O₂^- and OH", respectively, do not decrease the phototoxicity. The photodecomposition of Compound I was inhibited by sodium azide, enhanced by human serum albumin and unaffected by mannitol. Stable photoproducts of Compound I were not toxic to the cells. The quantum yield of singlet oxygen based on its emission at 1270 nm was 0.19 and 0.35 for Compound I and s2 ± 10^-3 and 10^-2 for piroxicam in D₂O and C₆H₆, respectively. While the extremely low quantum yield for singlet oxygen from piroxicam appears to account for its lack of phototoxicity, the phototoxicity mechanism for its metabolite, Compound I, most likely does involve singlet oxygen.     

REACTION OF SINGLET OXYGEN WITH CHOLESTEROL IN LIPOSOMAL MEMBRANES. EFFECT OF MEMBRANE FLUIDITY ON THE PHOTOOXIDATION OF CHOLESTEROL

Abstract— The photosensitized oxygenation of cholesterol in liposomal membranes results in the formation of the characteristic singlet oxygen product, 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide. The yield of this product was found to be strongly temperature dependent with 6- to 7-fold increase above the transition temperature of the membrane using 1,2-dipalmitoyl- sn -glycero-3-phosphorylcholine or 1.2-dimyristoyl- sn -glycero-3-phosphorylcholine. In contrast, the formation of the radical autoxidation products of cholesterol was not significantly enhanced in these systems by higher temperatures. We conclude from the results that the oxidation of cholesterol by singlet oxygen in the artificial membranes is affected by both membrane fluidity and amount of hematoporphyrin incorporated into the membrane. The ratio of cholesterol to phospholipid which affects the morphology of membranes is a crucial factor in determining the yield of the singlet oxygen product.     

THE USE OF SPECIFIC RADIOIMMUNOASSAYS TO DETERMINE ACTION SPECTRA FOR THE PHOTOLYSIS OF (6-4) PHOTOPRODUCTS

Abstract. A radioimmunoassay (RIA) was developed which specifically detects a photoproduct produced by the near-UV photolysis of pyrimidine(6-4)pyrimidone photoproducts. This assay was used in conjunction with a previously characterized RIA which specifically detects (6-4) photoproducts to determine the relative efficiency of wavelengths between 265 and 435 nm for photolysing these lesions. The rate of loss of antibody-binding sites associated with (6-4) photoproducts correlates with the production of those associated with its photolysis product. Action spectra for both the loss of (6-4) photoproducts and the induction of the photolysis product parallel the absorption spectrum of the (6-4) photoproduct.     

SINGLET OXYGEN GENERATION BY FUROCOUMARIN TRIPLET STATES—I. LINEAR FUROCOUMARINS (PSORALENS)

Abstract— Triplet extinction coefficients and hence singlet → triplet intersystem crossing quantum yields have been measured in benzene for a number of linear furocoumarins including pseudopsoralen, 5, 8-dimethoxypsoralen, 4, 5', 8-trimethylpsoralen and 3-carbethoxypseudopsoralen. These triplet yields were then used in conjunction with the corresponding quantum yields of singlet oxygen formation, measured in oxygenated solution, to estimate the fractions of furocoumarin triplets which when quenched by ground state oxygen produce singlet excited oxygen, similar data being obtained for psoralen, 5-methoxypsoralen, 8-methoxypsoralen and 3-carbethoxypsoralen. The superoxide anion radical was not detected from these oxygen quenching reactions, nor was a contribution to the singlet oxygen yield found from furocoumarin excited singlet state quenching by oxygen. The fraction of furocoumarin-oxygen quenching interactions leading to singlet oxygen varied between 0.13 (for 5, 8-dimethoxypsoralen) and unity (for 3-carbethoxypsoralen), and thus needs to be taken into account, as well as the triplet quantum yields, in assessing photobiological processes involving singlet oxygen.     

ACTION SPECTRA FOR SUPEROXIDE GENERATION AND UV AND VISIBLE LIGHT PHOTOAVOIDANCE IN PLASMODIA OF Physarum polycephalum

Abstract— The initial photochemical process leading to photoavoidance by plasmodia of an albino strain of Physarum Plasmodium was studied. Superoxide (O), detected as superoxide dismutase (SOD)-inhibitable electron spin resonance (ESR) signal of a spin trap (tBN), was formed upon irradiation. The amount of O formed increased linearl) with log fluence rate above the threshold. The photoavoidance to radiation at wavelengths between200–800 nm also showed the similar linear relationship in log fluence rate-response curves. Thresholds for photoavoidance and O generation agreed with each other and the action spectra showed peaks at about 260, 370, and 460 nm. Thus, active oxygen generated by photosensitization seems to trigger the UV and blue light photoavoidance.     

HYDROGEN PEROXIDE-DEPENDENT GENERATION OF SINGLET MOLECULAR OXYGEN BY HUMAN SALIVA: ITS DETECTION BY CHEMILUMINESCENCE FROM A Cypridina LUCIFERIN ANALOG

By the addition of hydrogen peroxide to human saliva, chemiluminescence from Cypridina luciferin analog (CCLA) and oxygen evolution were observed. Chemiluminescence was inhibited by inhibitors of salivary peroxidase, azide and cyanide and by a singlet oxygen quencher, crocin. Deuterium oxide (99.75%) stimulated the initial increase of CCLA by15–50% and the integrated CCLA 2.1-3.6-fold. The result suggest that the generation of singlet oxygen by peroxidase in human saliva depends on hydrogen peroxide.     

ANOXIC PHOTODAMAGE IN THE PRESENCE OF PORPHYRINS: EVIDENCE FOR THE LACK OF EFFECTS ON MITOCHONDRIAL MEMBRANES

Abstract When isolated mitochondria are irradiated in the presence of 50 μg/ml hematoporphyrin or hematoporphyrin derivative in the respiratory medium, the irradiation is at least twenty-fold less effective in impairing the Ca²⁺ pump in the absence of oxygen than in its presence. This result suggests that photosensitization by non oxygen-depending pathways has few or no effects on cell membranes, a result somewhat at variance with those observed with liposomes.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN BY AMINES-III. EVIDENCE FOR A CHARGE-TRANSFER-LIKE COMPLEX

Abstract— A series of amines were found to quench singlet oxygen in the order tertiary > secondary > primary, with a reasonable correlation between the log of their rate constant of quenching and their ionization potential. In addition, a Hammett rho plot gave a rho value of - 1.39 for the quenching of singlet oxygen by a series of substituted N, N-dimethylanilines, in good agreement with the results obtained by a different method. It was found that some of the amines (anilines) quenched the triplet state of the dye-sensitizer (Rose Bengal) used for the production of singlet oxygen. Corrections in the results were made in the calculations of rates of quenching of singlet oxygen to allow for the triplet-state quenching. No extensive quenching of the singlet state of the dye was observed at the concentrations of the amines necessary for singlet-oxygen quenching. In one case (N, N, N', N'-tetramethylphenylenediamine) there was no observable chemical reaction between singlet oxygen and the amine. It was concluded that singlet oxygen undergoes physical quenching by the amines via partial charge-transfer intermediates.     

THE UV ACTION SPECTRA FOR THE CLONE-FORMING ABILITY OF CULTURED HUMAN MELANOCYTES AND KERATINOCYTES

Abstract Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone-forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22-1.92 for the UV-C and UV-R wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV-A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV-C and UV-B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV-A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV-A light.