Effect of structural modifications on photosensitizing activities of hypocrellin dyes: EPR and spectrophotometric studies.

Mono-substituted hypocrellin B (MHB) and di-substituted hypocrellin B (DHB) by mercaptoacetic acid are new photosensitizers synthesized to improve the red absorption and water solubility of the parent hypocrellin B (HB). The photochemistries (Type I and/or Type II) of MHB and DHB have been studied in homogeneous solutions using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic homogeneous DMSO solution, DHB*- (or MHB*-) was predominantly photoproduced via self-electron transfer between the excited- and ground-state species. The presence of an electron donor significantly promotes the formation of the reduced form of DHB (or MHB). As compared with hypocrellin B, the efficiencies of DHB*- and MHB*- generation was enhanced obviously. When oxygen-saturated solutions of DHB (or MHB) were illuminated with 532 nm light, singlet oxygen (1O2), superoxide anion radical (O2*-), hydroxyl radical (*OH) and hydrogen peroxide (H2O2) were formed. DHB and MHB generate 1O2 with quantum yields of 0.18 and 0.22, respectively, which are much lower than that of HB (0.76) in chloroform. The superoxide anion radical was significantly enhanced by the presence of electron donors. The rate of O2*- production was also dependent on the concentration of DHB or MHB. Moreover, O2*- upon disproportionation can generate H2O2 and ultimately the highly reactive *OH via the Fenton reaction and other pathway with the involvement of DHB*- (or MHB*-). As in the case of DHB*- (or MHB*-), the efficiencies of O2*- and *OH generation by DHB and MHB were also enhanced obviously, consistent with the fact that DHB*- (or MHB*-) acts as the precursor of O2* and thus *OH. These findings suggest that the photodynamic actions of DHB and MHB may proceed via enhanced Type I mechanism and reduced Type II mechanism as compared with that of HB.     

ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

Photodynamic action of C-phycocyanins obtained from marine and fresh water cyanobacterial cultures: a comparative study using EPR spin trapping technique

C-phycocyanins, major biliproteins of blue green algae (cyanobacteria), widely used as colourants in food and cosmetics are known for their antioxidant as well as therapeutic potential. Recent claims indicating phycobiliproteins exert stronger photodynamic action on tumor cells than clinically approved hematoporphyrin derivatives motivate us to investigate the photodynamic action of two newly isolated C-phycocyanins from Phormidium [PHR] and Lyngbya [LY] spp, respectively in comparison with known C-phycocyanin from Spirulina sp. [SPI]. Photolysis of air saturated solutions of PHR, LY and SPI in the presence of 2,2,6,6-Tetramethyl piperidinol (TEMPL) generated three line EPR spectrum characteristic of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxyl (TEMPOL). The increase in intensity of the EPR spectrum with time of irradiation and decrease in intensity, in the presence of 1O2 quencher DABCO confirm the formation of 1O2. Photoirradiation in the presence of spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) generated EPR signal characteristic of O2(-) adduct. Efficiency of 1O2 generation is of the order LY > PHR> SPI. The yield of reactive oxygen species (ROS) generation is found to be 1O2>O2(-) indicating type II mechanism to be the prominent pathway for photosensitation by phycocyanins.     

Synthesis and EPR investigations of new aminated hypocrellin derivatives

Hypocrellins are novel photodynamic agents. A recent advance in the synthesis of hypocrellin congeners results in the production of two amino-substituted hypocrellin B derivatives in high yield via photochemical reaction. Both compounds exhibit similar photodynamic activity as hypocrellin B in terms of type I and type II mechanisms. In anaerobic media, semiquinone anion radicals can be detected by electron paramagnetic resonance (EPR) under irradiation; while superoxide anion radical, hydroxyl radical and singlet oxygen are photoproduced when oxygen was present. The quantum yields of singlet oxygen by these two new compounds are determined to be 0.72 and 0.64, respectively, similar to that of hypocrellin B. The comparison of the photosensitization chemistry of compounds 1 and 2 in liposomes with that in homogeneous solution has also been made. In liposomes, the type II photoprocess was favored and predominant over the type I photoprocess due to the decreased interactions between dye molecules. Both compounds exhibit much stronger red light absorption than the parent hypocrellin and therefore, merit investigation as photosensitizers.     

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

Photosensitized formation of ascorbate radicals by chloroaluminum phthalocyanine tetrasulfonate: an electron spin resonance study.

The chloroaluminum phthalocyanine tetrasulfonate sensitized photooxidation of ascorbic acid to ascorbate radical (A.-) was followed by electron spin resonance (ESR) spectroscopy. In air saturated aqueous media, steady-state amounts of A.- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished--more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN3 and tryptophan) was studied, as was replacement of water by D2O and saturation with O2. The results are indicative of free radical production by direct reaction between ascorbate ion and sensitized phthalocyanine (a Type I mechanism) in competition with the (Type II) reaction of HA- with singlet oxygen, a reaction which does not produce ascorbate radical intermediates.     

Butylamino-demethoxy-hypocrellins and photodynamic therapy decreases human cancer in vitro and in vivo.

2-Butylamino-2-demethoxy-hypocrellin A (BAHA) and B (BAHB) are new photosensitizers synthesized by a mild reaction of hypocrellins and butylamine. In BAHA and BAHB, the peri-hydroxylated perylenequinone structure of the parent hypocrellins is preserved and the red absorption is enhanced distinctly. Electron paramagnetic resonance spin trapping measurements and 9,10-diphenylanthracene bleaching studies were used to investigate the photodynamic action of BAHA and BAHB in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2(*-)) produced by illuminating BAHA and BAHB in aerobic solution have been observed. Compared with hypocrellin A and B, BAHA and BAHB primarily remained able to generate 1O2 and enhanced distinctly the O2(*-)-generating abilities. The photodynamic action of BAHA and BAHB in the therapy of cancer was investigated in vitro and in vivo. Both in vitro and in vivo results revealed a significant decrease in cancer cell growth. Laser or dye alone had no effect, indicating that intratumor BAHA and laser therapy may prove useful in unresectable cancer.     

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Cytotoxicity, redox cycling and photodynamic action of two naturally occurring quinones

Two naturally occurring anthraquinones, barleriaquinone-I (BQ-I) and barleriaquinone-II (BQ-II), extracted from Barleria buxifolia, are tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma cells (MCF7). Cytotoxicities, measured as LD(50) (50% inhibition of colony formation) values, show BQ-II to be more active than BQ-I. Electron paramagnetic resonance studies confirm that BQ-II is reductively activated by NADH:cytochrome c reductase to superoxide anion radical. Cyclic voltammetric studies show one quasi-reversible redox couple for both BQ-I and BQ-II. Also, aerobic solutions of both BQ-I and BQ-II on visible illumination generate reactive oxygen species. Formation of O*-2 is studied by both EPR spin trapping and SOD-inhibitable cytochrome c reduction techniques. BQ-I generates more singlet oxygen as evidenced from the photobleaching of N,N-dimethyl-4-nitrosoaniline.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.     

The effects of singlet oxygen produced by photodynamic action on the mitochondrial permeability transition differ in accordance with the localization of the sensitizer

We have examined whether the effects of singlet oxygen (1O2) produced by photodynamic action on the mitochondrial permeability transition (PT) can be modulated by the localization of photosensitizers in irradiated mitochondria. We have previously shown that oxidation due to 1O2 photogenerated in hematoporphyrin (HP)-loaded mitochondria can prevent opening of the PT pores, likely after degradation of some critical histidines (Salet et al, 1997, J. Biol. Chem. 272, 21938-21943). Equally, in the present study we have irradiated mitochondria in the presence of a structurally different photosensitizer producing 1O2, namely 4,5',8-trimethylpsoralen (TMP). Fluorescence studies show that TMP binds to protein sites which differ from those of HP. In sharp contrast with HP, TMP-driven photodynamic action triggers per se pore opening. Interestingly, this inducing effect is inhibited when TMP-treated mitochondria are irradiated after addition of mersalyl, a specific reagent protecting thiol groups of the inner mitochondrial membrane that are oriented toward the external hydrophilic phase. This fact suggests that 1O2-mediated thiol oxidation is responsible for TMP-photoinduced pore opening. Taken together, these findings suggest that 1O2 can activate or inactivate a cellular function such as mitochondrial PT depending on the site where it is produced in the mitochondrial membrane.     

Activated oxygen species produced by photoexcited furocoumarin derivatives

Furocoumarin derivatives (FCD) are investigated in order to determine their ability to photosensitize the production of activated oxygen species. Using the method based on the specific singlet oxygen (1O2) oxydation of cholesterol, all FCD except bergaten appeared to be able to produce 1O2 with various efficiencies. EPR spin trapping experiments show that photoexcited FCD produce hydroxyl radicals as detected by the formation of a DMPO-OH signal which can be abolished when the photosensitization reaction is carried out in the presence of specific OH scavengers. Moreover, the photo-ejection of hydrated electron (e-) by FCD is demonstrated by the loss of paramagnetic absorption of nitroxide free radicals as e- trap.     

Photosensitized damage to calf thymus DNA by a hypocrellin derivative: mechanisms under aerobic and anaerobic conditions

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB*- radical), followed by the second electron transfer from DNA to DCHB*- or the disproportionation of DCHB*- to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.     

Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (lambda>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.