Porphyrin depth in lipid bilayers as determined by iodide and parallax fluorescence quenching methods and its effect on photosensitizing efficiency

Photosensitization by porphyrins and other tetrapyrrole chromophores is used in biology and medicine to kill cells. This light-triggered generation of singlet oxygen is used to eradicate cancer cells in a process dubbed "photodynamic therapy," or PDT. Most photosensitizers are of amphiphilic character and they partition into cellular lipid membranes. The photodamage that they inflict to the host cell is mainly localized in membrane proteins. This photosensitized damage must occur in competition with the rapid diffusion of singlet oxygen through the lipid phase and its escape into the aqueous phase. In this article we show that the extent of damage can be modulated by employing modified hemato- and protoporphyrins, which have alkyl spacers of varying lengths between the tetrapyrrole ring and the carboxylate groups that are anchored at the lipid/water interface. The chromophore part of the molecule, and the point of generation of singlet oxygen, is thus located at a deeper position in the bilayer. The photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. The vertical insertion of the sensitizers was assessed by two fluorescence-quenching techniques: by iodide ions that come from the aqueous phase; and by spin-probe-labeled phospholipids, that are incorporated into the bilayer, using the parallax method. These methods also show that temperature has a small effect on the depth when the membrane is in the liquid phase. However, when the bilayer undergoes a phase transition to the solid gel phase, the porphyrins are extruded toward the water interface as the temperature is lowered. These results, together with a previous publication in this journal, represent a unique and precedental case where the vertical location of a small molecule in a membrane has an effect on its membranal activity.     

Genotoxicity of singlet oxygen

Singlet oxygen, ¹O₂(¹Δ_g), fulfills essential prerequisites for a genotoxic substance, like hydroxyl radicals and other oxygen radicals: it can react efficiently with DNA and it can be generated inside cells, e.g. by photosensitization and enzymatic oxidation. As might be anticipated from the non-radical character of singlet oxygen, the pattern of DNA modifications it produces is very different from that caused by hydroxyl radicals. While hydroxyl radicals produce DNA strand breaks and sites of base loss (AP sites) in high yield and react with all four bases of DNA, singlet oxygen generates predominantly modified guanine residues and few strand breaks and AP sites. There is now convincing evidence that a major product of base modification caused by singlet oxygen is 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). Indeed, the recently reported miscoding properties of 8-hydroxyguanine can explain the predominant type of mutations observed when DNA modified by singlet oxygen is replicated in cells. There are also strong indications that singlet oxygen generated by photosensitization can act as an ultimate DNA modifying species inside cells. However, indirect genotoxic mechanisms involving other reactive oxygen species produced from singlet oxygen are also possible and appear to predominate in some cases. The cellular defense system against oxidants consists of effective singlet oxygen scavengers such as carotenoids. The observation that carotenoids can inhibit neoplastic cell transformation when administered not only together with but also after the application of chemical or physical carcinogens might indicate a role of singlet oxygen in tumor promotion that could be independent of the direct or indirect DNA damaging properties.     

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes

An effect of β-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15 mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.     

Singlet oxygen photosensitization by EGFP and its chromophore HBDI

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 μs in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the β-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.     

Studies on the photochemical and photocytotoxic properties of the new PDT photosensitizer aluminum sulfonated phthalocyanine.

The properties of photosensitization of sulfonated aluminum phthalocyanine (ALSPC), a new photosensitizer of potential use in cancer photodynamic therapy (PDT) was studied on both the molecular and cellular levels. The mechanism of ALSPC photosensitization on the molecular level was investigated by testing its efficiency of singlet oxygen (1O2) production, using the method of tryptophan degradation and that of ESR spectroscopy and observing the enhancing effect of D2O and the quenching effect of NaN3. Results of all these experiments confirmed the important role of the Type II or 1O2 mechanism in ALSPC photosensitization. In our in-vitro experiments, ALSPC's incorporation into cells and its photocytotoxic effect were investigated on a human liver cancer cell line. The cell incorporation was illustrated by the laser-excited fluorescence spectra emitted both from cell homogenate and cell monolayers incubated with ALSPC aqueous solution. The position of fluorescence peak observed, implied that ALSPC exists in the cells mainly as monomers. The efficiency of cell killing of ALSPC photosensitization was estimated by counting surviving cells with the method of trypan blue staining and by the method of radioisotope labelling. Experiments using the latter method also showed DNA damage caused by ALSPC photosensitization.     

Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores

To gain insight into mechanisms of photodynamic modification of biological membranes, we studied an impact of visible light in combination with a photosensitizer on translocation of various substances across artificial (vesicular and planar) bilayer lipid membranes (BLMs). Along with induction of carboxyfluorescein leakage from liposomes, pronounced stimulation of lipid flip-flop between the two monolayers was found after photosensitization, both processes being prevented by the singlet oxygen quencher sodium azide. On the contrary, no enhancement of potassium chloride efflux from liposomes was detected by conductometry under these conditions. Illumination of planar BLMs in the presence of a photosensitizer led to a marked increase in membrane permeability to amphiphilic 2-n-octylmalonic acid, but practically no change in the permeability to ammonia, which agreed with selective character of the photosensitized leakage of fluorescent dyes from liposomes (Pashkovskaya et al., Langmuir, 2010). Thus, the effect on transbilayer movement of molecules elicited by the photodynamic treatment substantially depended on the kind of translocated species, in particular, on their lipophilicity. Based on similarity with results of previous electroporation studies, we hypothesized about photodynamic induction of "pre-pores" or "hydrophobic defects" permeable to amphiphilic compounds and less permeable to hydrophilic substances and inorganic ions.     

某些物理化学因素对竹红菌甲素敏化的人红细胞膜乙酰胆碱酯酶的光失活作用的影响

本文以人红细胞膜乙酰胆碱酯酶力作用对象,研究了甲素浓度、pH、温度等因素对甲素致敏的酶光失活的影响,并计算了不同条件下的酶失活速率常数.甲素与某些光敏化剂相比,有以下特点:(1)甲素光敏化效率随着pH降低而增加,(2)光强指数α>1,(3)甲素在400nm—600nm波长范围内均有较大的光敏化作用.后性氧清除剂的试验结果表明,乙酰胆碱酯酶的光失活主要是单线态氧的作用,其它活性氧也有一定作用.     

Interaction of all-<Emphasis Type="Italic">trans</Emphasis>-Retinal with bilayer lipid membranes

The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3: 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen.     

In situ evidence for the involvement of superoxide anions in cutaneous porphyrin photosensitization

Dihematoporphyrin ether, also known as Photofrin-II (Pf-II) is currently used in the diagnosis and management of a variety of epithelial neoplasms, in a modality known as photodynamic therapy (PDT). A major drawback of these porphyrins for PDT is their ability to evoke prolonged cutaneous photosensitization. The mechanism of tumor ablation and cutaneous photosensitization by these photosensitizers is thought to relate to the generation of one or more reactive oxygen species such as superoxide anion, singlet oxygen and hydroxyl radical. However, the role of these oxygen species has not been established unequivocally. In this study, the mechanism of Pf-II-mediated cutaneous photosensitization was examined using murine ear swelling as a marker. The mice treated with Pf-II and light demonstrated two-fold enhancement of ear swelling whereas animals treated with the SOD mimic, beta-carotene and dimethyl sulfoxide (DMSO) had considerably less ear swelling (p less than 0.01). The observed protective effect was dependent on the dose of each quencher and followed the pattern SOD mimic DMSO beta-carotene. The histopathologic alterations caused by Pf-II photosensitization were significantly alleviated by pretreatment with SOD mimic whereas beta-carotene and (DMSO) were less effective. Inhibitors of superoxide dismutase (sodium diethyldithiocarbamate) and catalase (hydroxyl amine and 3, amino 1,2,4-triazole) augmented Pf-II-mediated cutaneous photosensitization. These data provide the first in vivo evidence for the involvement of superoxide anion in cutaneous porphyrin photosensitization.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Enzymatically generated electronically excited molecules induce transformation of 4-thiouridine to uridine

Oxidation of 4-thiouridine-5'-monophosphate in t-RNA, from E. coli, by singlet oxygen generated via self-sensitization, photosensitization or by energy transfer from bioenergized systems yields uridine-5'-monophosphate. Studies with absorption, fluorescence and circular dichroism techniques showed similar interactions between singlet oxygen and the nucleotide in t-RNA generated by either optical or enzymatic systems. Protection by histidine and an enhancement of the photodegradation in the presence of D2O corroborates the important role of singlet oxygen in these processes.     

Molecular mechanisms of photosensitization

The first part of this article is devoted to basic concepts of photosensitization and to the primary photophysical and photochemistry processes involved in the reaction. The electronic configuration of molecular oxygen in its ground or activated states, which intervene in numerous photosensitized reactions, is reviewed. Finally, the main photosensitized reactions are reviewed and classified into three different groups: reactions due to radicals (type I), reactions due to singlet oxygen (type II) and those which do not involve oxygen (type III).     

Synthetic control of interchromophoric interaction in cationic bis-porphyrins toward efficient DNA photocleavage and singlet oxygen production in aqueous solution

We have synthesized cationic bis-porphyrins and their zinc(II) complexes with two TMPyP-like chromophores bridged by p- or m-xylylenediamine to develop effective DNA photocleaving agents. The xylylene linkers and zinc ion were introduced to control interchromophoric interaction that should be involved in photosensitization of the cationic bis-porphyrins. The molar absorptivities of all the bis-porphyrins in aqueous solution remained unchanged over a wide range of concentrations, indicating the absence of self-aggregation property. In particular, the molar absorptivity of the zinc(II) complex of the p-xylylenediamine-linked bis-porphyrin in aqueous solution was 2.0 times as large as that of unichromophoric ZnTMPyP, suggesting the absence of both intermolecular and intramolecular interchromophoric interaction. The metal-free p-xylylenediamine-linked bis-porphyrin showed the more efficient conversion ability of supercoiled to nicked circular pUC18 plasmid DNA by photosensitization than the metal-free m-xylylenediamine-linked one. Furthermore, the zinc complexes of the bis-porphyrins exhibited the more potent DNA photocleavage than did the metal-free bis-porphyrins. Singlet oxygen productivity of the four cationic bis-porphyrins was determined by measuring the decomposition rate of 1,3-diphenylisobenzofuran. The amount of singlet oxygen generated by photosensitization of the zinc(II) complex of the p-xylylenediamine-linked bis-porphyrin in aqueous solution was 2.1 times as large as ZnTMPyP, indicating the full singlet oxygen productivity. A significant relationship between the DNA photocleaving abilities and the singlet oxygen productivities of the cationic porphyrins in aqueous solution was found. Hence, the degree of the intramolecular interchromophoric interaction, the DNA photocleaving ability, and the singlet oxygen productivity of the cationic bis-porphyrins in aqueous solution were successfully controlled by means of the introduction of the appropriate linker and metal ion.     

L-ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: generalized antioxidant property of vitamin C

L-ascorbic acid quenches singlet (1 delta g) molecular oxygen in aqueous media (pH 6.8 for [1H]H2O and pD 7.2 for [2H]D2O) as measured directly by monitoring (0,0) 1 delta g leads to 3 sigma-g emission at 1.28 micron. Singlet oxygen was generated at room temperature in the solutions via photosensitization of sodium chrysene sulfonate; this sulfonated polycyclic hydrocarbon was synthesized to provide a water soluble chromophore inert to usual dye-ascorbate photobleaching. A marked isotope effect is found; kHQ2O is 3.3 times faster than kDQ2O, suggesting ascorbic acid is chemically quenching singlet oxygen.     

Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii

In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms.     

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.     

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.     

Reactivity of singlet molecular oxygen with cholesterol in a phospholipid membrane matrix. A model for oxidative damage of membranes

Photooxidation of cholesterol in liposomes with hematoporphyrin sensitization has been studied. With liposomal samples in which the hematoporphyrin is incorporated in the membrane, the yield of the characteristic singlet oxygen product, 3β-hydroxycholest-6-ene 5α-hydroperoxide, was approximately 6 times greater than that observed in the samples in which the hematoporphyrin was outside the membrane. Small amounts of 3β-hydroxycholest-5-ene 7α- and 7β-hydroperoxides, radical autoxidation products, were formed in both samples. Photolysis of a dispersion of cholesterol in an aqueous solution of hematoporphyrin gave no singlet oxygen products. It is concluded from these results that endogenous singlet oxygen when formed in the phospho-lipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; whereas exogenous singlet oxygen generated outside the membrane is quenched by solvent before appreciable diffusion into the membrane can occur.     

Use of pyrene as a luminescence indicator of the viscosity of model and biological membranes

The pyrene movement in a lipid bilayer has been shown to occur not only in the lateral but also transmembrane direction. Within the excited state lifetime the pyrene monomer elevates from the depth to the polar regions of the membrane and emits a luminescence photon. The excimer does not exhibit any marked transmembrane movement while luminescing from the hydrophobic regions. The luminescence quenching efficiency of monomers and excimers depends on the depth of quencher penetration into the membrane. In the lipid bilayer the pyrene luminescence is strongly quenched by molecular oxygen. The pyrene binding to membrane proteins protects it from quenching. A conclusion has been made that the carrying out estimations of membrane viscosity from pyrene luminescence require considerable correction.     

Reactive oxidizing species produced near the plasma membrane induce apoptosis in bovine aorta endothelial cells

Many cytotoxic agents initiate apoptosis by generating reactive oxidizing species (ROS). The goal of this study was to determine whether apoptosis could be induced by initial reactions of ROS near the plasma membrane. Bovine aorta endothelial cells (BAEC) were illuminated with evanescent wave visible radiation, which has limited penetration into the basal surface of cells, or by trans-radiation. Imaging of fluorescent dyes localizing in the plasma membrane, mitochondria, or nucleus confirmed that evanescent wave radiation excited only dyes in and near the plasma membrane. Singlet oxygen, an ROS generated by photosensitization, has a very short lifetime, ensuring that it oxidizes molecules residing in or very close to the plasma membrane when evanescent wave radiation is used. Cells with condensed nuclei were considered apoptotic and were quantified after treatment with varying doses of light. Annexin V staining without propidium iodide staining confirmed that these cells were apoptotic. The doses required to induce apoptosis using evanescent wave radiation were 10-fold greater than those needed for trans-irradiation. Quantitative analysis of the evanescent wave penetration into cells supports a mechanism in which the singlet oxygen created near the plasma membrane, rather than at intracellular sites, was responsible for initiation of apoptosis.