RIBOFLAVIN-PHOTOSENSITIZED OXIDATIVE DEGRADATION OF A VARIETY OF LIGNIN MODEL COMPOUNDS

Oxidation of several lignin model compounds with alkylated paraphenolic groups by photosensitizing riboflavin (RF). rose bengal (RB) and methylene blue (MB) was examined. Photosensitizing RF cleaved l–(3'-4'-diethoxyphenyl)-1.3 dihydroxy-2-(4-methoxyphenyl)propane (I). 4-ethoxy-3-methoxyphenylglycerol-(3-guaiacyl ether (II) and l-(4'-ethoxy-3'-methoxyphenyI)-1,3 dihydroxypropane (IV) at their respective C_α-C_β bonds. Riboflavin also oxidized 3.4-diethoxy-benzaldehyde (VI) to the corresponding acid, and hydroxylated the conjugated olefin l-(4'-ethoxy-3'-methoxyphenyl)1.2 propene (III) to yield the initial product IV. In contrast, MB and RB hydroxylated III but had no effect on I, II, IV or VI under identical conditions. This suggested RF effected transformations via a hydrogen radical abstraction (Type I) rather than a ¹O₂ mediated reaction.
To confirm this, the effects of deuterium oxide (D_:0) and oxygen pressure on the photosensitizing dye reactions were examined. The effect of D₂0 on tryptophan (Trp) oxidation and hydroxylation of III by MB and RB was significant, indicating involvement of ¹O₂. D₂O had no significant effect on cleavage of the diarylpropane (I) and the olefin (III) by RF, indicating these reactions did not proceed by a Type II mechanism. While O₂ pressure effect on Trp oxidation by MB was insignificant, it had a large negative effect on cleavage of I by RF. These results, coupled with the relatively slow oxidation rate of Trp by RF. indicate that photosensitizing RF produces ¹O₂ inefficiently and is not effecting oxidation of these lignin model compounds via a Type II mechanism.     

PHOTOSENSITIZED PRODUCTION OF SUPEROXIDE ANION BY MONOCHROMATIC (290–405 nm) ULTRAVIOLET IRRADIATION OF NADH and NADPH COENZYMES

Abstract— The reduced pyridine coenzymes NADPH and NADH produced superoxide anion("CK") from ground state molecular oxygen when irradiated by ultraviolet (UV) radiation extending from 290 to 405 nm as detected by cytochrome c reduction. Superoxide dismutase (SOD), but not catalase or heat-inactivated SOD, decreased the amount of cytochrome c reduced, indicating that O₂⁻ was responsible for the reduction of cytochrome c. Decreased oxygen tension during irradiation also inhibited production of O₂⁻. Quantum yields for the production of the anion were in the region of 10⁻⁷ to 10⁻⁹ mol per photon. These data indicate that NADH and NADPH can act as type II photosensitizers of both far-and near-UV radiation, and that the deleterious biological effects of exposure to these radiations such as erythema and dermal carcinogenesis may be mediated at least in part through the generation of O₂⁻.     

A COMPARATIVE STUDY OF PHOTODYNAMIC OXIDATION AND RADIOFREQUENCY-DISCHARGE-GENERATED 1O2 OXIDATION OF GUANOSINE

Abstract— Guanosine-2-¹⁴C and guanosine-8-¹⁴C were subjected, under similar environmental conditions, to oxidation either by radiofrequency-discharge-generated singlet oxygen or by a methylene-blue-sensitized photodynamic reaction. The reaction products in both cases were compared chromatographically. The product distributions were not identical, indicating differing modes of oxidation in these two systems. It is suggested that both Type I and Type II oxidation mechanisms are operative in the photodynamic system, whereas only Type II-like reactions occur in the radio-frequency-discharge system.     

PHOTOLYSIS OF AMIODARONE, AN ANTIARRHYTHMIC DRUG

Abstract— The photochemical behaviour of amiodarone was examined in vitro in order to get more insight on the chemical reactions involved in the cutaneous phototoxicity processes . Irradiation at 300 nm of amiodarone degassed in ethanol solution leads to a photodehalogenation followed by a much slower α-cleavage reaction. Desethylamiodarone, the main metabolite of AD was found to undergo the same reaction as AD. Results of photosensitization and quenching experiments together with phosphorescence spectra indicated that the reaction proceeds via the triplet excited stateof amiodarone. Radical species formed during photolysis were identified by ESR spectroscopy. CH₃CHOH, HO₂ and an unidentified radical were detected using 5,5-dimethyl-1-pyrroline-1-oxide as spin trap. In aerated solutions, photosensitization of oxygen by amiodarone was demonstrated by adding singlet oxygen scavengers such as dimethylfuran and cholesterol. Overall, these results suggest that Type I and Type II mechanisms may take place in the phototoxicity of amiodarone and its metabolite.     

竹红菌素类光动力药物*

天然竹红菌素（包括竹红菌甲素和乙素）作为一种新型的光动力抗肿瘤药物,与血卟啉衍生物(HpD)相比,具有单一和确定的化学组成、易纯化、暗毒性低、兼具Type I和Type II双重敏化机制等优点。本文综述了竹红菌素类光敏剂在光动力疗法领域中近五年来的最新研究进展,包括竹红菌素的化学修饰、物理包裹、与生物大分子的相互作用以及动物细胞的体外和体内光动力性质研究,并对竹红菌素类光敏剂未来的研究方向作了展望。     

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.     

MECHANISMS OF THE PHOTOSENSITIZED OXIDATION OF TYRAMINE

Abstract— …According to the criteria of enhancement in D₂O and inhibition by sodium azide, the oxidation of tyramine photosensitized by methylene blue is largely a singlet oxygen or Type II process. Its quantum yield approximates 0.3 in D₂O at pH 10. There is a less efficient reaction not quenched by azide, which is assigned to a dye-substrate or Type I process. It gives rise to products with distinct bands at 320 and 285nm. Products of the Type I reaction are further oxidized by singlet oxygen and thereby compete with tyramine for this reagent. Kinetic parameters were estimated by computer simulation of the dependence of quantum yield on extent of reaction. The rate constant for reaction of O₂ (¹Δ_g) with tyramine was estimated to be 2.8 × 10⁸ M ^-1 s ^-1± 20% at pH 10. The reaction was also sensitized by hypericin in what appears to be a Type II process.     

REDUCED PHOTOINACTIVATION OF 10-DODECYL ACRIDINE ORANGE-SENSITIZED YEAST CELLS AT HIGH FLUENCE RATES MEASUREMENTS AND COMPUTER SIMULATIONS

Abstract-During the development of a photodamage cell sorter several photosensitizers were tested for their ability to photoinactivate more than 90% of the sensitized cells after a brief irradiation with a fluence of 10 kJ/m². In pilot experiments, yeast cells sensitized with 10-dodecyl acridine orange (DAO) were effectively photoinactivated after receiving a fluence of 10 kJ/m² delivered in 8 s. However, when the same fluence was delivered in 3 μ s during passage through a focused laser beam in the cell sorter, all cells survived.
Computer simulations of the relevant photophysical and chemical reactions inside the irradiated cell were used to investigate the cause of this phenomenon. The results indicated that the absence of photoinactivation by DAO, after flash irradiations, was caused by the combined effects of (1) limited oxygen diffusion into the cell and (2) a reduced number of collisions between photosensitizer triplet and oxygen molecules during the irradiation due to saturation of the intracellular photosensitizer triplet concentration. The contributions of triplet-triplet annihilation and triplet quenching by ground state photosensitizer molecules were found to be minimal and not significant. These findings indicate that Type II photosensitizers are incapable of rapid selective photoinactivation in cell sorters.     

PHOTOPRODUCTION OF HYDROGEN FROM LINKED CHROMOPHORES

Abstract On irradiation of solutions of anthryl-substituted cobalt(III) cage complexes, [(l-(anthryl-9-methylamino)-8-methyl-3,6,10,13,16,19-hexaazabicyclo [6.6.6] eicosane) cobalt(III)]³⁺ or [(l-(4-an-thryl-9)-3-aza-butyl-l-amino)-8-methyl-3,6,10,13,16,19-hexaazabicyclo [6.6.6] eicosane)cobalt (III)]³* in the presence of ethylenediaminetetraacetic acid and platinum catalysts hydrogen was produced. These complexes act as coupled photosensitizers (anthracene moiety) and electron relays (cobalt cage) to produce H₂ via energy trapping and intramolecular electron transfer initially. The intensity of fluorescence and the photochemical reactivity favour the latter complex and the excited singlet state of the anthracene chromophore is invoked as the intermediate state leading to the reduction of Co(III) to Co(II).     

Photosensitized DNA damage and its protection via a novel mechanism

UVA, which accounts for approximately 95% of solar UV radiation, can cause mutations and skin cancer. Based mainly on the results of our study, this paper summarizes the mechanisms of UVA-induced DNA damage in the presence of various photosensitizers, and also proposes a new mechanism for its chemoprevention. UVA radiation induces DNA damage at the 5'-G of 5'-GG-3' sequence in double-stranded DNA through Type I mechanism, which involves electron transfer from guanine to activated photosensitizers. Endogenous sensitizers such as riboflavin and pterin derivatives and an exogenous sensitizer nalidixic acid mediate DNA photodamage via this mechanism. The major Type II mechanism involves the generation of singlet oxygen from photoactivated sensitizers, including hematoporphyrin and a fluoroquinolone antibacterial lomefloxacin, resulting in damage to guanines without preference for consecutive guanines. UVA also produces superoxide anion radical by an electron transfer from photoexcited sensitizers to oxygen (minor Type II mechanism), and DNA damage is induced by reactive species generated through the interaction of hydrogen peroxide with metal ions. The involvement of these mechanisms in UVA carcinogenesis is discussed. In addition, we found that xanthone derivatives inhibited DNA damage caused by photoexcited riboflavin via the quenching of its excited triplet state. It is thus considered that naturally occurring quenchers including xanthone derivatives may act as novel chemopreventive agents against photocarcinogenesis.     

Probe-Free Direct Identification of Type I and Type II Photosensitized Oxidation Using Field-Induced Droplet Ionization Mass Spectrometry

While Type I and Type II photosensitizers are often carefully tailored to achieve their respective advantages in treating different cancers, the identifications of the Type I and II mechanisms as such, the key reaction intermediates, and the consequent oxidation products of the substrates have never been easy. Using our unique home-built field-induced droplet ionization mass spectrometry (FIDI-MS) method that selectively samples molecules at the air–water interface, here we show the facile determination of both Type I and II mechanisms of a poster-child photosensitizer, temoporfin, without the addition of any probes. The unstable doublet radical resulting from the hydrogen abstraction by the triplet temoporfin through the Type I mechanism is captured, manifesting the in situ advantage of FIDI-MS. We anticipate that the method developed in this study can be widely utilized in the future designs of novel photosensitizers and the screening of their photosensitization mechanisms.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

CHEMILUMINESCENCE OF SOME REACTIONS WITH MOLECULAR OXYGEN

Abstract— Many inorganic oxidation reactions involving a variety of oxidizing agents show chemiluminescence in the spectral region of 400–600 mp. Excited O₂O₂-associates are assumed to be the emitting molecules formed by bimolecular recombination of HO _x O₂-radicals. Oxidation of sodium sulfite with molecular oxygen in the presence of Cu- and Fe-basic oxides shows chemiluminescence which originates from the reactions of the heavy metal complexes of OH- and O, H-radicals with O₂. The simplest way of producing a radical from O₂ is by the uptake of one electron. Suitable electron donors, such as hydro-quinones and semiquinones, emit light if treated with oxygen. In certain organic solvents OH- can also act as electron donor. Its presence causes the formation of semiquinones from quinones in the absence of oxygen. The chemiluminescence which is observable upon treatment of alkaline dimethylsulfoxide, dioxane, pyridine-water and alcohol-water mixtures with oxygen is also attributable to electron transfer from OH- to O₂.     

Preclinical Assessment of Hypocrellin B and Hypocrellin B Derivatives as Sensitizers for Photodynamic Therapy of Cancer: Progress Update

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-( N,N -dimethylami-no)-propylamine-HB]. Extinction coefficients at 630 nm (φ₆₃₀) are 6230, 6190 and 4800, respectively; and ¹O₂ quantum yields, φ, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMToVEd tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhi-murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.     

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.     

SELECTIVE TYPE I PHOTOOXIDATIONS IN MIXTURES OF PORPHYRINS AND ELECTROPHILIC NITROIMIDAZOLES

Abstract— Electrophilic compounds metronidazole (METRO) and misonidazole (MISO), considerably enhance the photooxidation quantum yield of Type I photodynamic substrates such as Trp, Tyr and Cys. For the latter, the quantum yield of photooxidation which can be much greater than one, suggests radical chain reactions. On the other hand, METRO and MISO inhibit ¹O₂ formation because they react at diffusion controlled rate (∼10⁹ M⁻¹ S⁻¹) with porphyrin triplets forming, porphyrin radical cations. As a result, the porphyrins are still able to photosensitize the destruction of Type I substrates even in the absence of O₂. These results are discussed with respect to the possibility of increasing the sensitivity of hypoxic cells to porphyrin photosensitization.     

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen‐dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen (¹O₂ ¹?_g) by energy transfer to molecular oxygen. In addition, superoxide anion radical () can be generated by a charge‐transfer reaction involving O₂ or more likely indirectly as the result of O₂‐mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. can also undergo dismutation into H₂O₂, the precursor of the highly reactive hydroxyl radical () that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one‐electron oxidation and singlet oxygen reactions.     

TEMPORAL and SPECTRAL SEPARATION OF SINGLET OXYGEN LUMINESCENCE FROM NEAR INFRARED EMITTING PHOTOSENSITIZERS

Abstract— The luminescence emission of singlet molecular oxygen (¹O₂) generated by bacteriopheophytin a, a near-infrared-emitting photosensitizer, was measured using a new high-sensitivity spectrometer system for time- and spectral-resolved near-infrared detection. The instrument uses a low energy pulsed nitrogen laser (40 μJ per pulse) to excite the photosensitizer optically and is capable of a time resolution of 40 ns per data point and an instrument response function of 350 ns FWHM (full width at half maximum). The use of a low-energy (and relatively low cost) source provides sufficient system sensitivity to measure time-resolved spectra in the near infrared with high spectral and temporal resolution. The simultaneous detection, with high accuracy and repeatability, of both the temporal and spectral dependence of the photoprocesses of ¹O₂ generation, especially with near-infrared-emitting photosensitizers, may further stimulate the current intensive investigations concerning the activity of ¹O₂ to biomolecules.     

Effect of Oxygen Concentration on Photo-oxidation and Photosensitizer Bleaching in Butter

The effect of headspace oxygen concentration and color of light on photo-oxidation and degradation of photosensitizers in butter was studied. Butter samples were stored under 0%, 0.4%, 0.8%, 1.6%, 3.0%, 5.0%, 21% oxygen, and exposed to violet, green or red light. Storage time was 36 h. Degree of photo-oxidation was measured by a trained sensory panel. Photobleaching of six different photosensitizers was estimated based on front face fluorescence excitation and emission landscapes and spectral curve resolution (parallel factor analysis). The higher oxygen concentration, the more sensory degraded were the samples. Violet light resulted in slightly higher degrees of photo-oxidation than green and red light for low oxygen concentrations. Bleaching rate and course as function of O₂ concentration differed between the photosensitizers. It is suggested that the rate of photobleaching is a balance between type I and type II photoreactions.