Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.     

MEASUREMENT OF FORMAL POTENTIALS FOR HYPERICIN IN DIMETHYLSULFOXIDE

The formal potentials for hypericin are measured in dimethylsulfoxide using cyclic voltammetry. Two reversible one-electron reductions and a more poorly defined multielectron oxidation are observed. The formal potentials for these processes are consistent with the visible spectrum of hypericin and they indicated that hypericin is both a good oxidizing agent and a good reducing agent in the excited state.     

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.     

Photophysical properties of Hypericum perforatum L. extracts--novel photosensitizers for PDT

We report the preparation of the methanolic extract (ME), and polar methanolic fraction (PMF) from the plant Hypericum perforatum L. The extracts contain various photosensitizing constituents such as naphthodianthrone derivatives (in 1.37% w/w), and chlorophylls (in 0.08% w/w). Upon light emission these constituents can be activated, providing photodynamic properties to the extracts, and making them a potent, new class, natural photosensitizers for use in photodynamic therapy (PDT), and photodynamic diagnosis (PDD). The absorbance spectra of the extracts are similar to the spectrum of hypericin, the main naphthodianthrone identified within, with two major bands at 548 and 590 nm. The fluorescence spectra in ethanol exhibit two main bands around 595 and 640 nm, in accordance with the spectrum of pure hypericin. The fluorescence intensity of PMF at 595 nm is only eight times less than the intensity of pure hypericin at the same wavelength, even though its hypericin concentration is only 0.57% w/w. The dependence of the PMF fluorescence signal on the pH of the medium, alone and in comparison with the signal of hypericin, has been investigated. PMF signal fades steadily, and smoothly both in acidic, and basic environment.     

Hypericin derivatives: substituent effects on radical-anion formation

The electron-transfer properties of the hypericin derivatives, dibromo-, hexaacetyl-, hexamethyl- and desmethylhypericin, were studied. Cyclovoltammetric measurements revealed that dibromo- and desmethylhypericin have almost the same redox potentials as the parent hypericin. Substitution of the hydroxyl groups by acetoxy leads to less negative E1/2 values, whereas methoxy substitution induces more negative values. Electron paramagnetic resonance (EPR)/electron nuclear double resonance/general TRIPLE spectroscopy and quantum mechanical calculations were used to establish the structure of the one-electron reduced stages of hypericin derivatives. Proton loss in the bay region, already demonstrated for hypericin, was also found for dibromo- and desmethylhypericin. The spin and charge of the radical ions are predominately confined to the central biphenoquinone moiety of the hypericin skeleton. Generation of the radical ions by in situ electrolysis indicates that the redox potentials of hypericin, dibromo- and desmethylhypericin, containing hydroxyls at the 1, 3, 4, 6, 8 and 13 positions, largely depend on the solvent. With phosphate-buffered saline (pH 7.4)/dimethylsulfoxide (DMSO) as the solvent the EPR spectra of the corresponding radical ions appear at markedly lower potentials than in pure DMSO and N,N'-dimethylformamide. However, this effect is not observable for hexaacetyl- and hexamethyl-hypericin-lacking hydroxyl groups. In all cases the EPR data and calculations revealed the presence of 7,14 tautomers.     

RESONANCE RAMAN AND SURFACE-ENHANCED RESONANCE RAMAN SPECTROSCOPY OF HYPERICIN

Hypericin has been found to exhibit a variety of photodynamic effects. To correlate biological activity with molecular structure, complete physical characterization of hypericin is required. The vibrational spectrum has been determined and resonance Raman and surface enhanced resonance Raman scattering spectra are reported. In addition, the Raman spectrum of a model compound has been studied to facilitate assignment of the vibrational modes of hypericin.     

Photophysics and multifunctionality of hypericin-like pigments in heterotrich ciliates: a phylogenetic perspective

In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.     

Maristentorin, a novel pigment from the positively phototactic marine ciliate Maristentor dinoferus, is structurally related to hypericin and stentorin

The photoreceptor pigment of the heterotrich ciliate, Maristentor dinoferus, has been characterized. It is structurally similar to those of Stentor coeruleus and Blepharisma japonicum but differs significantly in that it bears no aromatic hydrogens. The structure of the pigment, maristentorin, is based upon the hypericin skeleton, and its spectra are nearly identical to those of hypericin but shifted toward the red. Within experimental error, its fluorescence lifetime is identical to that of hypericin, approximately 5.5 ns in dimethylsulfoxide. It is remarkable that while the pigments are structurally similar in S. coeruleus and M. dinoferus, in the former there is an abrupt photophobic response, whereas in the latter there is a slow response toward light. The roles of the hypericin-like pigments in the heterotrich ciliates are discussed as potentially analogous in Maristentor.     

Conformational studies by dynamic NMR. 74.(1) stereomutations of the conformational enantiomers in peri-substituted 1-acylnaphthalenes

Naphthalenes bearing an acyl and a phenyl group in a peri relationship give rise to a pair of enantiomers in the temperature range where the rotations of the acyl group are slow. Such enantiomers were observed by means of low temperature NMR spectra in chiral environments. The barrier to rotation for the acyl substituents, that causes the interconversion of the enantiomers, was demonstrated to be lower than that for the phenyl group. In an appropriately synthesized derivative it was possible to measure the two barriers that were found equal to 10.4 and 15.9 kcal mol(-)(1), respectively. The barriers for the acyl group rotation increase regularly (from 9.5 to 13.2 kcal mol(-)(1)) with the increasing dimension of the RCO groups (R = Me, Et, Pr(i), Bu(t)). When a bromine atom replaces the phenyl group, the enantiomerization barrier for the corresponding acyl derivatives increases significantly.     

Skin photosensitization with topical hypericin in hairless mice

Hypericin, a naturally occurring photosensitizer, exhibits interesting in vitro photobiological activities, which suggest that the compound is a potential antipsoriatic agent. In this study, the possibility of hypericin penetrating the skin in photo-active concentrations has been studied. Hypericin is incorporated in either emulsifying ointment supplemented with solketal (hypericin content: 0.05%) or in polyethylene glycol (PEG) ointment (hypericin content: 0.5%) and applied to the skin of hairless mice for 4 h. After removing excess ointment, the mice are then irradiated with different light doses using a 500 W halogen lamp. As a positive control, intraperitoneally (i.p.) administered hypericin (10 and 40 mg/kg) has also been tested. Erythema, desquamation and erosions are demonstrated in the mice treated with hypericin in emulsifying ointment with solketal using a light dose of at least 4.5 J/cm2. In general, these reactions correlate well with those of i.p. administered hypericin (40 mg/kg), indicating that hypericin incorporated in emulsifying ointment with solketal is well absorbed by the skin of the mice. However, for the i.p. administered hypericin (40 mg/kg), we could not evaluate phototoxic reactions in the group of animals that received a light dose of 108 J/cm2, as they all died 12-24 h after irradiation, indicating extreme photosensitization with systemic hypericin at higher light doses. On the contrary, there is no measurable skin photosensitivity induced by hypericin when incorporated in PEG ointment or when 10 mg/kg hypericin is i.p. administered. Our results show that hypericin incorporated in a suitable vehicle can be delivered to the skin in photo-active concentrations. Using a vehicle such as emulsifying ointment with solketal, it will be possible to explore the photo-activity of hypericin in the treatment of psoriasis and other skin diseases.     

Chiral discrimination in the 13C and 2H NMR of the crown and saddle isomers of nonamethoxy-cyclotriveratrylene in chiral liquid-crystalline solutions

We report 2H and 13C NMR spectra of the crown and saddle isomers of nonamethoxy-tribenzocyclononene (1), dissolved in lyotropic achiral and chiral liquid-crystalline solutions based on poly-gamma-benzyl-glutamate and poly-gamma-benzyl-L-glutamate (PBG and PBLG). The 2H-[1H] measurements include spectra of compound 1 deuterated in the ring methylene and in the aromatic sites as well as of the methyl groups in natural abundance. Carbon-13 spectra were recorded in natural abundance as well as in two isotopomers enriched in the ring methylene and one of the methoxy groups. The crown isomer (c-1) is rigid with C3 symmetry and can be separated into its enantiomers using a chiral high-performance liquid chromatography column. The NMR spectra of racemic c-1 in PBLG solutions exhibit two sets of lines due to the enantiomers. The peaks were identified by comparing the spectra with those of the neat enantiomers. Analysis of the 2H quadrupolar splittings and the 13C residual chemical shift anisotropies shows that the dominant factor determining the chiral discrimination is the difference in the ordering of the two enantiomers in the chiral liquid crystals. The saddle isomer (s-1) is highly flexible, undergoing fast pseudorotation between six conformers. The "frozen" conformers have C1 symmetry and are therefore chiral. Three of these comprise one enantiomer, and the other three the second one. However, the rapidly interconverting species has, on the average, a C3h symmetry and is therefore achiral. The methylene groups in the latter are, however, prostereogenic, and their hydrogen/deuterium-carbon bonds constitute enantiotopic pairs. The 2H NMR spectra of the s-1 methylene-deuterated in PBLG solutions exhibit, in fact, enantio-discrimination with two quadrupolar doublets. This is in contrast to rigid prochiral molecules with a threefold symmetry axis, which normally do not show such discrimination. A detailed analysis of the effect is presented, and it is argued that the discrimination observed for s-1 reflects the different ordering of its enantiomers during the pseudorotation cycle.     

PHOTODYNAMIC ACTION IN Stentor coeruleus SENSITIZED BY ENDOGENOUS PIGMENT STENTORIN

Abstract— Stentorin acts as the photoreceptor for the step-up photophobic and negative phototactic responses in Stentor coeruleus . The chromophore of stentorin appears to be hypericin which is linked to apoprotein. In addition to the photomovement responses of the organism, S. coeruleus was found to be photodynamically sensitive to light absorbed by the hypericin chromophore, as the apparent action spectrum for the photodynamic killing matches the absorption spectrum of stentorin. The protective effect of β-carotene and crocetin on the photodynamic killing of S. coeruleus suggests that singlet oxygen generated by the stentorin-sensitization plays an important role, according to the so-called Type II mechanism of photosensitization. The generation of singlet oxygen via hypericin triplet was confirmed by in vitro photooxidation of tryptophan as a substrate. The photodynamic killing was more effective in deuterium oxide than in H₂O in both the photosensitization by stentorin (endogenous) and added hypericin (exogenous). These results are consistent with the involvement of singlet oxygen in the photodynamic killing of S. coeruleus .     

Lowest excited triplet states of hypericin and isohypericin

The fluorescence and phosphorescence of hypericin and isohypericin were studied in an ethanol matrix at 1.2 K. The prompt fluorescence spectra are mirror images of the absorption around the 0-0 transition, as expected. The 0-0 vibronic lines of the phosphorescence are found at 13190 and 13622 cm⁻¹, and the phosphorescence decay times are 2.79 and 6.6 ms at 1.2 K for hypericin and isohypericin respectively. The fluorescence excitation spectrum of isohypericin reveals a small trace of a different pigment, possibly a tautomeric form of isohypericin. The decay of the phosphorescence of isohypericin is biexponential. The slow component is attributed to an unknown contaminant with a high quantum yield of phosphorescence.     

PHOTOACTIVATION OF HYPERICIN GENERATES SINGLET OXYGEN IN MITOCHONDRIA AND INHIBITS SUCCINOXIDASE

Photosensitized inhibition of mitochondrial succinoxidase by hypericin was measured in vitro and found to be drug-dose, light-dose, and wavelength dependent. Singlet oxygen generation, monitored using the singlet oxygen trap tetramethylethylene, and oxygen consumption in isolated mitochondria sensitized by hypericin were also light-dose and wavelength dependent. Unequivocal evidence for the generation of singlet oxygen was obtained using kinetic isotope ratios of products from the reaction between singlet oxygen and geminally deuterated tetramethylethylene. An action spectrum for the inhibition of succinoxidase was measured at wavelengths between 400 and 700 nm and found to parallel the recorded visible absorption spectrum of hypericin in isolated mitochondria. The greatest singlet oxygen generation, oxygen consumption, and succinoxidase inhibition occurred with white light or 600 nm irradiation. These data are consistent with a type II singlet-oxygen-mediated mechanism for hypericin induced photosensitized inhibition of mitochondrial succinoxidase.     

Reduction in hypericin-induced phototoxicity by Hypericum perforatum extracts and pure compounds

Clinical evidence suggests that administration of Hypericum perforatum (Hp) extracts containing the photo-activated hypericin compounds may cause fewer skin photosensitization reactions than administration of pure hypericin. This study was conducted to determine whether the phototoxicity of hypericin in HaCaT keratinocytes could be attenuated by H. perforatum extracts and constituents. Two extracts, when supplemented with 20 microM hypericin: (1) an ethanol re-extraction of residue following a chloroform extraction (denoted ethanol(-chloroform)) (3.35 microM hypericin and 124.0 microM total flavonoids); and (2) a chloroform extract (hypericin and flavonoids not detected), showed 25% and 50% (p<0.0001) less phototoxicity than 20 microM hypericin alone. Two H. perforatum constituents, when supplemented with 20 microM hypericin: (1) 10 microM chlorogenic acid; and (2) 0.25 microM pyropheophorbide, exhibited 24% (p<0.05) and 40% (p<0.05) less phototoxicity than 20 microM hypericin alone. The peroxidation of arachidonic acid was assessed as a measure of oxidative damage by photo-activated hypericin, but this parameter of lipid peroxidation was not influenced by the extracts or constituents. However alpha-tocopherol, a known antioxidant also did not influence the amount of lipid peroxidation induced in this system. These observations indicate that hypericin combined with H. perforatum extracts or constituents may exert less phototoxicity than pure hypericin, but possibly not through a reduction in arachidonic acid peroxidation.     

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups

The ¹H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD₃OH solutions indicate significantly deshielded signals in the region of 14-15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional ¹H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14-15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV-vis and LC/MS measurements.     

Cellular photodestruction induced by hypericin in AY-27 rat bladder carcinoma cells

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.     

Enantioselective analysis of pheniramine in urine by charged CD-mediated CZE provided with a fiber-based DAD and an on-line sample pretreatment by capillary ITP

Application potentialities of CZE on-line coupled with capillary ITP and DAD to the identification and determination of trace concentration levels (microg/L) of pheniramine (PHM) enantiomers and their metabolites present in complex ionic matrices of biological origin (urine) are shown. An enhanced (enantio)selectivity of the CZE separation system obtained by the addition of carboxyethyl-beta-CD (CE-beta-CD) to the carrier electrolyte provided CZE conditions for a reliable identification of similar/identical DAD spectra of structurally related compounds (PHM enantiomers and their metabolites) in clinical urine samples differing in qualitative and quantitative composition of sample matrix constituents. A high sample loadability (a 30 microL sample injection volume), partial sample clean-up (removing macroconstituents from the sample), and preconcentration of the analytes in ITP stage resulted in the decrease of concentration LOD for PHM enantiomers in urine to 5.2 and 6.8 microg/L (2.2 x 10(-8) and 2.8 x 10(-8) mol/L), without using any sample pretreatment technique. The background correction and smoothing procedure applied to the raw DAD spectra provided analytically relevant DAD spectra of PHM enantiomers and their metabolites also when they were present in urine sample (30 microL injection volumes of ten-times diluted urine sample) at a 9 x 10(-) (8) mol/L concentration. DAD spectra of PHM enantiomers present in urine samples matched their reference spectra with reasonable certainties. DAD spectra of PHM metabolites were compared with the reference spectra of PHM enantiomers and a good match was found which indicates the similarities in the structures of enantiomers and their metabolites detected in the urine samples. This fact allows performing the quantitative analyses of PHM metabolites in the urine samples by applying the calibration parameters of PHM enantiomers also for PHM metabolites and the results show the possibilities of using the ITP-CZE-DAD combination for the direct analysis of PHM enantiomers and/or their metabolites in urine without any sample pretreatment. ITP-CZE-DAD method with oppositely charged selector is suggested to use in clinical research as it provides favorable performance parameters including sensitivity, linearity, precision, recovery, and robustness with minimal demands on sample preparation.     

Synthesis of hydroxy and methoxy perylene quinones, their spectroscopic and computational characterization, and their antiviral activity

Hydroxy and methoxy perylene quinones are synthesized in an attempt to isolate the essential spectroscopic and biological features of light-induced antiviral agents such as hypericin and hypocrellin. Unlike their naturally occurring counterparts, these synthetic quinones bear the carbonyl, hydroxyl, and methoxy groups in the "bay region." The hydroxy and methoxy compounds have rich absorption spectra with broad features in the visible (approximately 450-800 nm) and relatively more intense and narrow features at wavelengths < or = 350 nm. High-level ab initio quantum mechanical calculations assign the features in the absorption spectra to electronic transitions from S0 to S2 and to higher-lying electronic states. The calculations indicate that in the ground state the trans dihydroxy isomer is 12.5 kcal/mol lower in energy than the cis dihydroxy isomer and is thus the only species present. The lowest-energy trans methoxy ground state isomer and the lowest-energy cis methoxy ground state isomer are found to be degenerate. An additional cis methoxy isomer 6.3 kcal/mol higher in energy than the global minimum is assumed to contribute to the spectrum and is also considered. Finally, the synthetic compounds exhibit similar light-induced antiviral activity to each other, but significantly less than that of hypericin.     

"Competitive quenching": a mechanism by which perihydroxylated perylenequinone photosensitizers can prevent adverse phototoxic damage caused by verteporfin during photodynamic therapy

Incorporation of photodynamic therapy into clinical practice for induction of vascular photo-occlusion highlights the need to prevent adverse phototoxicity to sensitive juxtaposed tissues, particularly in the retina. We developed a system termed "competitive quenching" to prevent adverse phototoxic damage. It involves differential compartmentalization of a photoactivator to the intravascular compartment for photoexcitation and delivery of phototoxicity to targeted vessels. A different photodynamic agent is partitioned to the extravascular retinal space to quench reactive oxygen species generated by photosensitization, thereby protecting the adjacent retinal tissues from adverse phototoxicity. The absorption spectra of quenchers must span wavelengths that are shorter and excluded from the spectral range of photoexcitation light to prevent photoactivation of the quencher. Perihydroxylated perylenequinones were found to be suitable to function as "competitive quenchers" with the prototype hypericin identified as a potent quencher. Here we examined the mechanisms operative in competitive quenching and suggest that hypericin forms a complex with verteporfin, thereby quenching singlet oxygen formation. Furthermore, we show that hypericin, with six phenolic hydroxyls, protects retinal and endothelial hybridoma cells from phototoxicity more effectively than the dimethyl tetrahydroxy helianthrone structural analog with only four such phenolic hydroxyls. The findings suggest that hydroxyl numbers contribute to the efficacy of competitive quenching.