Theoretical investigation of the photosensitization mechanisms of urocanic acid

The photosensitization mechanisms of urocanic acid (UA), the main skin chromophores of ultraviolet (UV) light, are investigated by means of theoretical calculations. The results indicate that the direct photooxidative damage to DNA bases by triplet state UA through electron transfer reaction is not favorable on thermodynamic grounds. However, UA can photogenerate various reactive oxygen species (ROS, e.g., (1)O(2), O(2)(-)) theoretically and the ROS-generating mechanisms are illustrated as follows. Firstly, the (1)O(2)-generating pathway involves direct energy transfer between triplet state UA and (3)O(2). Secondly, UA gives birth to O(2)(-) through two pathways: (i) direct electron transfer between triplet state UA and (3)O(2); (ii) electron transfer between anion radical of UA (generated through autoionization reactions) and (3)O(2).     

Characteristics of the primary reaction of hypocrellin A photosensitization

The primary reaction of photosensitization of Hypocrellin A (HA) has been studied by using techniques of ESR, spin-trapping and spin-counteraction. The experiments show that HA is able to generate not only 1O2, but also O-2, .OH and HA- which are observed for the first time. The conversion of generating active oxygen into generating nonoxygen free radical is confirmed as well. Based on the characteristics of the primary reaction which generates these active substances (transient products), it is proposed that the photosensitized damage for the biological system by HA is probably related to not only 1O2 itself, but also a multiple effect from 1O2 as well as O-2, .OH and HA- free radical.     

Singlet molecular oxygen quenching ability of carotenoids in a reverse-micelle membrane mimetic system

The influence of the medium heterogeneity upon the bimolecular rate constants for the physical quenching, kq, and chemical quenching, kr, of singlet molecular oxygen O2(1deltag) by seven natural and three synthetic carotenoids (CAR) with different substituent patterns was studied in a reverse micelle system of sodium bis(2-ethylhexyl)sulfosuccinate, hexane and water. Because O2(1deltag) was generated inside the water pools of the reverse micelles by photosensitization of the water-soluble dye rose bengal and the CAR are mainly located in the external hexane pseudophase, the quenching process was interpreted using a pseudophase model for the partition of 02(1deltag) between the water pools and the organic pseudophases. The kq values were mainly dependent on the extent of the double-bond conjugation of the CAR, as demonstrated by a good empirical relationship between log(k(q)) and the energy E(S) of the longest wavelength transition pi-->pi* of the CAR. In contrast, the kr values were almost independent of the extent of the double-bond-conjugated system and about four orders of magnitude lower than kq. However, in all cases, CAR photobleaching was observed with the formation of various oxidation products, depending on the photosensitization time. Chromatographic and spectroscopic product analysis for the reaction products of beta-carotene with O2(1deltag) indicated the formation of the beta-carotene-5,8-endoperoxide as the primary oxidation product.     

Protease-triggered photosensitizing beacon based on singlet oxygen quenching and activation

We report a new concept for type-II photosensitization, based on incorporating the photosensitizer (PS) and a singlet-oxygen (1O2) quenching/scavenging molecule onto a disease-targeting linker, such that the PS becomes activatable by light only when targeting has occurred. In this first proof-of-concept report, a model photosensitizing beacon was synthesized containing a pyropheophorbide as the PS and a carotenoid as the 1O2 quencher. These were kept in close proximity by the self-folding of a caspase-3-specific peptide sequence. Upon caspase-3-induced cleavage, the 1O2 production increased markedly, as measured directly by 1O2 near-infrared luminescence and lifetime measurements.     

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (³H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium-neon (HeNe) laser and TBO was investigated by using deuterium oxide (D₂O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L-tryptophan. There were 9.0 log₁₀ and 2 log₁₀ reductions in the presence of D₂O and H₂O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm²), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm²) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre-gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.     

PHOTOSENSITIZED DAMAGE TO SUPERCOILED PLASMID DNA INDUCED BY 334-nm RADIATION IN THE PRESENCE OF 2-THIOURACIL CONSISTS OF ALKALI- and PIPERIDINE-LABILE SITES AS WELL AS FRANK STRAND BREAKS

A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, gamma-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48, 607-613] produced much different proportions, with a PLS:FSB ratio of 4.1:1. These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.     

Role of Nuclear Factor-KB in Colon Cancer Cell Apoptosis Mediated by Aminopyropheophorbide Photosensitization

Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4',6-diamidine-2'-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-kappa B is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-kappa B turned out to be involved in an antiapoptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-kappa B alpha was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.     

Photosensitization by anticancer agents. 11. Mechanisms of photosensitization of human leukemic cells by diaminoanthraquinones: singlet oxygen and radical reactions.

The synthesis of several aminoanthraquinone derivatives (AAQs), designed to suppress the dark toxicity and to promote more efficient cancer cell photosensitization for potential use in photodynamic therapy (PDT), is described. The following AAQs were synthesized: 1-NH2-4,5-(MeO)2-AQ (1), 1,5-(NH2)2-4,8-(MeO)2-AQ (2), 1,8-(NH2)2-4,5-(MeO)2-AQ (3), and 1,5-(NHPhMe)2-4,8-(MeO)2-AQ (8). The agents exhibit strong absorption in the region 480-620 nm. Possible mechanisms of photosensitization were studied by measuring 1O2 phosphorescence at 1270 nm, detecting superoxide radicals employing an electron paramagnetic resonance (EPR)-spin trapping technique, and measuring oxygen consumption during the photo-oxidation of a representative biological electron donor, NADH. Strong phosphorescence from 1O2 was observed upon illumination of 2 and 3 in C6H6 (quantum yield of 0.25 and 0.5 respectively), and in EtOH (quantum yield of 0.23 and 0.34). The 1-amino-AQ (1) was the weakest 1O2 sensitizer, with quantum yield of 0.13 in benzene. No phosphorescence was observed in EtOH. A superoxide radical was detected as a spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in irradiated benzene solutions of 1, 2 or 3 and DMPO. AAQs 2 and 3 sensitized photo-oxidation of NADH in H2O/EtOH mixture with the intermediacy of singlet oxygen as judged by the effect of sodium azide on the photostimulated oxygen consumption. Evolution of O2 upon addition of catalase to the illuminated solution confirmed the ultimate formation of hydrogen peroxide. These findings suggested that the (di)amino-dimethoxyanthraquinones might exert photosensitization via both Type I and Type II mechanisms. The AAQs were tested for their ability to photosensitize K562 human chronic myeloid leukemic cells in culture. Viability was measured using the 3,4,5-diethylthiazol-2,5-diphenyl tetrazolium blue assay, and DNA and possible membrane damage were assessed. The results from illuminating cells with light > 475 nm show that for the 1,5-compounds, the presence of methoxy substituents at 4,8 positions reduces the dark toxicity from ID50 of 23 to 250 microM and for the 1,8-compounds correspondingly from ID50 of 53 to > 300 microM. In the 1,5-series this decrease of the dark toxicity is accompanied by an increase in light-induced dose modification from 8.85 to 14.4. Differences exist in the mechanisms of cytotoxicity between the prototype phenolic AAQs and their methoxy counterparts. It appears that the cytotoxic action of the latter causes cell damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks.(ABSTRACT TRUNCATED AT 400 WORDS)     

Photodegradation and photosensitization of mycosporine-like amino acids

The photodegradation and photosensitization of several mycosporine-like amino acids (MAAs) were investigated. The photodegradation of the MAA, palythine, was tested with three photosensitizers: riboflavin, rose bengal and natural seawater. For comparison of degradation rates, the riboflavin-mediated photosensitization of six other MAAs was also examined. When riboflavin was used as a photosensitizer in distilled water, MAAs were undetectable after 1.5h. Palythine showed little photodegradation when rose bengal was added as the photosensitizer (k=0.12x10(-3)m(2)kJ(-1)). Palythine dissolved in natural seawater containing high nitrate concentrations also showed slow photodegradation rate constants (k=0.26x10(-3)m(2)kJ(-1)) over a 24-h period of constant irradiation. Similar experiments in deep seawater with porphyra-334 and shinorine resulted in 75% of the initial MAA remaining after 4h of irradiation and rates of 0.018 and 0.026x10(-3) m(2) kJ(-1), respectively. Experiments conducted in deep seawater with riboflavin additions resulted in photodegradation rate constants between 0.77x10(-3) and 1.19x10(-3)m(2)kJ(-1) for shinorine and porphyra-334, respectively. Photoproduct formation appeared to be minimal with the presence of a dehydration product of the cycloheximine ring structure indicated as well as the presence of amino acids. Evidence continues to build for the role of MAAs as potent and stable UV absorbers. This study further highlights the photostability of several MAAs in both distilled and seawater in the presence of photosensitizers.     

Triplet formation of 4-thiothymidine and its photosensitization to oxygen studied by time-resolved thermal lensing technique

Excited-state dynamics of 4-thiothymidine (S4-TdR) and its photosensitization to molecular oxygen in solution with UVA irradiation were investigated. Absorption and emission spectra measurements revealed that UVA photolysis of S4-TdR gives rise to a population of T1(pipi*), following S2(pipi*) --> S1(npi*) internal conversion. In transient absorption measurement, the 355 nm laser photolysis gave broad absorption (380-600 nm) bands of triplet S4-TdR. The time-resolved thermal lensing (TRTL) signal of S4-TdR containing the thermal component due to decay of triplet S4-TdR was clearly observed by the 355 nm laser excitation. The quantum yield for S1 --> T1 intersystem crossing was estimated to be unity by a triplet quenching experiment with potassium iodide. In the presence of molecular oxygen, the photosensitization from triplet S4-TdR gave rise to singlet oxygen O2 (1Deltag) with a quantum yield of 0.50. Therapeutic implications of such singlet oxygen formation are discussed.     

Direct optical detection of singlet oxygen from a single cell

Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1deltag --> X3sigmag-) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.     

Triplet excited state characters and photosensitization mechanisms of alpha-terthienyl: a theoretical study

The triplet excited (T(1)) state characters of alpha-terthienyl (alpha-T) have been investigated using density functional theory calculations, based on which, its photosensitization mechanisms were explored. Primarily, the direct oxidation to the DNA bases by the T(1) state alpha-T through the electron transfer is not thermodynamically feasible. Secondly, 1O2 can be photogenerated both in benzene and water through the direct energy transfer from the T(1) state alpha-T to 3O2, while O2(.-) can only be formed in water through the electron transfer from the T(1) state alpha-T or alpha-T(-) to 3O2.     

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.     

Photosensitization of 2'-deoxyadenosine-5'-monophosphate by pterin

UV-A radiation (320-400 nm) induces damages to the DNA molecule and its components through photosensitized reactions. Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitization of 2'-deoxyadenosine-5'-monophosphate (dAMP) by pterin (PT) in aqueous solution under UV-A radiation. The effect of pH was evaluated, the participation of oxygen was investigated and the products analyzed. Kinetic studies revealed that the reactivity of dAMP towards singlet oxygen (1O2) is very low and that this reactive oxygen species does not participate in the mechanism of photosensitization, although it is produced by PT upon UV-A excitation. In contrast, analysis of irradiated solutions by means of electrospray ionization mass spectrometry strongly suggested that 8-oxo-7,8-dihydro-2'-deoxyadenosine-5'-monophosphate (8-oxo-dAMP) was produced, indicating that the photosensitized oxidation takes place via a type I mechanism (electron transfer).     

The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.     

Two-photon singlet oxygen microscopy: the challenges of working with single cells

A microscope is described in which singlet molecular oxygen, O2(a1deltag), is produced in a femtoliter focal volume via a nonlinear two-photon photosensitized process, and the 1270 nm phosphorescence from this population of O2(a1deltag) is detected in a photon counting experiment. Although two-photon excitation of a sensitizer is less efficient than excitation by a one-photon process, nonlinear excitation has several distinct advantages with respect to the spatial resolution accessible. Pertinent aspects of this two-photon O2(a1deltag) microscope were characterized using bulk solutions of photosensitizers. These data were compared to those obtained from a single biological cell upon linear one-photon excitation of a sensitizer incorporated in the cell. On the basis of the results obtained, we outline the challenges of using nonlinear optical techniques to create O2(aldeltag) at the single cell level and to then optically detect the O2(aldeltag) thus produced in a time-resolved experiment.     

Mechanistic and kinetic aspects of photosensitization in the presence of oxygen

Determining whether the first step of photooxygenation is Type I or Type II is a necessary prerequisite in order to establish the mechanism of photodynamic action. But this distinction is not sufficient, because other processes, both consecutive and competitive, commonly participate in the overall mechanism. Thus, in both Type I and Type II reactions, the initial products are often peroxides that can break down and induce free radical reactions. These aspects of photosensitization are discussed and illustrated by sensitizer/substrate systems involving (1) only radical reactions (decatungstate/alkane) and (2) reactions of singlet oxygen occurring in competitive and consecutive processes and possibly followed by radical reactions (methylene blue/2'-deoxyguanosine). Two other previously investigated systems involving, respectively, a Type II interaction followed by radical processes (methylene blue/alkene) and Type II reactions, some of which being competitive or consecutive (rose bengal/alkene), are briefly reconsidered.     

Versatile photosensitization system for 1O2-mediated oxidation of alkenes based on nafion-supported platinum(II) terpyridyl acetylide complex

[structure: see text] Platinum(II) terpyridyl acetylide complex (1) was incorporated into Nafion membranes as a photosensitizer, and the Nafion was immersed in an aqueous or organic solution of 7-dehydrocholesterol, alpha-pinene, or cyclopentadiene. This photosensitizer system can generate singlet oxygen ((1)O(2)) in high quantum yield to oxidize the alkenes in the solution outside the Nafion and can be easily removed from the reaction vessel at the end of the photooxidation.     

在光动力光疗中类卟啉稀土配合物光敏作用原初反应的特征: 产生自由基I型机制的ESR研究

本文用ESR方法研究了类卟啉稀土配合物[(CO2H-APPC)Gd]Cl2的光敏反应。用4-hydro-tetramethylpiperidine-N-oxide radical(4-hydro-TEMPO)作探针, 通过对其消自旋的作用, 证实[(CO2H-APPC)Gd]Cl2光敏反应中有阳离子自由基[(CO2H-APPC)Gd]^+产生, 加入还原剂可促使[(CO2H-APPC)Gd]^+生成。经由5, 5-Dimethyl-1-pyrrolineN-oxide(DMPO)对超氧阴离子(O2^-)和羟基自由基(.OH)的自旋捕捉及对该自旋加合物[DMPO-O2^-]和[DMPO-OH]的ESR测定, 证实有O2^-和.OH产生, 并用SOD清除O2^-和甲酸钠清除.OH的实验, 进一步证实O2^-和.OH的产生。上述结果说明[(CO2H-APPC)Gd]Cl2光敏反应存在着产生[(CO2H-APPC)Gd]^+和活性氧自由基的I型机制。     

Potential Involvement of Both Type I and Type II Mechanisms in M13 Virus Inactivation by Methylene Blue Photosensitization

We have investigated the mechanism of virus photoinactivation with methylene blue (MB) by conducting deuterium oxide (D₂O), azide ion (N₃^-) and oxygen-dependent, studies. Inactivation of M13 bacteriophage and singlet oxygen (¹O₂) generation by MB photosensitization were irradiation dose dependent. Inactivation of M13 was enhanced by D₂O and inhibited by N₃^-, suggesting that ¹O₂ participates in M13 inactivation by MB photosensitization. However, N₃^- did not inhibit M13 inactivation completely. On the other hand, deoxygenating the reaction solution still caused 52-67% of M13 inactivation observed in the presence of oxygen. These results suggest that ¹0₂-mediated (Type II) and sensitizer-mediated (Type I) reactions may both play roles in M13 inactivation by MB photosensitization.