Micellar effect on the scavenging of singlet molecular oxygen by hydroxybenzenes

The antioxidative effectiveness of three hydroxyaromatic derivatives (OHAD), namely phenol, resorcinol and phloroglucinol, as reflected by their scavenging ability of the photochemically generated species singlet molecular oxygen [O(2)((1)Delta(g))], was studied in aqueous solution and micellar media. Kinetic results, obtained through time-resolved phosphorescence detection of O(2)((1)Delta(g)) emission and polarographic methods, at pH 7 and 12 in homogeneous and CTAB micellar media, were compared. Calculated photooxidation quantum efficiencies (phi(r)), ranging from very low values ( approximately 0.01) to relatively high ones (approximately 0.3), depend on the preferential solubilization sites of the hydroxyaromatic derivatives either in the micellar or homogeneous media. OHAD in water pH 7 and in CTAB 0.02 M pH 12 exhibit their highest antioxidative efficiency as O(2)((1)Delta(g)) scavengers. In these conditions, they present the best degree of self protection against O(2)((1)Delta(g))-mediated photooxidation with relatively high values for the overall quenching rate constant and, simultaneously, the lowest phi(r).     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

Biological hydroperoxides and singlet molecular oxygen generation

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen ((1)O(2)) in biological systems. Recently, we have clearly demonstrated the generation of (1)O(2) in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of (1)O(2) in these reactions was the use of an isotopic labeled hydroperoxide, the (18)O-labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of (1)O(2) light emission. Using this approach we have observed the formation of (18)O-labeled (1)O(2) by chemical trapping of (1)O(2) with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC-MS/MS. The generation of (1)O(2) was also demonstrated by direct spectral characterization of (1)O(2) monomol light emission in the near-infrared region (lambda = 1270 nm). In summary, our studies demonstrated that LOOH can originate (1)O(2). The experimental evidences indicate that (1)O(2) is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation (1)O(2). This hypothesis is currently being investigated in our laboratory.     

Quenching of singlet molecular oxygen by screening pigments-- melanins and ommochromes

Synthetic DOPA-melanin and natural screening pigments--sepiomelanin and ommochromes are shown to quench the luminescence of singlet molecular oxygen (1O2) in aqueous (D2O, pD = 7.5-8.1) solutions. The rate constants of 1O2 quenching are found to be equal to (1.2 +/- 0.6) 10(8) M-1 s-1 for monomeric units in DOPA-melanin and to (3 +/- 1) 10(6) M-1 s-1 for ommochromes. The data suggest that screening is not the only function of melanins, which may play a role of inhibitors of photodynamic damage in living tissues.     

Studies on the singlet oxygen scavenging mechanism of human macular pigment

It is thought that direct quenching of singlet oxygen and scavenging free radicals by macular pigment carotenoids is a major mechanism for their beneficial effects against light-induced oxidative stress. Corresponding data from human tissue remains unavailable, however. In the studies reported here, electron paramagnetic resonance (EPR) spectroscopy was used to measure light-induced singlet oxygen generation in post-mortem human macula and retinal pigment epithelium/choroid (RPE/choroid). Under white-light illumination, production of singlet oxygen was detected in RPE/choroid but not in macular tissue, and we show that exogenously added macular carotenoids can quench RPE/choroid singlet oxygen. When the singlet oxygen quenching ability of the macular carotenoids was investigated in solution, it was shown that a mixture of meso-zeaxanthin, zeaxanthin, and lutein in a ratio of 1:1:1 can quench more singlet oxygen than the individual carotenoids at the same total concentration.     

Using the singlet oxygen scavenging property of carotenoid in photodynamic molecular beacons to minimize photodamage to non-targeted cells.

We recently introduced the concept of photodynamic molecular beacons (PMB) for selective control of photodynamic therapy (PDT). The PMB consists of a peptide linker that is sequence specific to a cancer-associated protease. A photosensitizer (PS) and a singlet oxygen (1O2) quencher are conjugated to the opposite ends of this linker. Proximity of the PS and quencher can efficiently inhibit 1O2 generation. In the presence of a targeted protease, the substrate sequence is cleaved and the PS and quencher will separate so that the PS can be photo-activated. There are two ways to optimize the PMB selectivity to cancer cells. The first is to increase the protease specificity to targeted cells and the second is to minimize the phototoxicity of intact (uncleaved) PMBs in non-targeted (normal) cells. Carotenoids (CARs) are well known in nature for their role in quenching excited states of PS and in directly scavenging 1O2. The purpose of this study is to evaluate whether the CAR with dual quenching modes (PS excited states deactivation and 1O2 scavenging) can be used to minimize the photodamage of intact PMBs to non-targeted cells. Thus, we synthesized a beacon (PPC) with a caspase-3 cleavable peptide linking a PS and a CAR quencher. It was confirmed that CAR deactivates the PS excited states and also directly scavenges 1O2. Moreover, the in vitro PDT response showed that CAR completely shuts off the photodynamic effect in non-targeted HepG(2) cells, while PS without CAR (control) remains highly potent even at a much lower (30-fold) dose.     

Myeloperoxidase singlet molecular oxygen generation detected by direct infrared electronic emission

Myeloperoxidase in micromolar concentrations reacting with half-millimolar stock solution H2O2 in acetate buffer containing KBr and in 50% D2O (pH + pD = 4.5) at 298 K is shown to generate singlet delta molecular oxygen efficiently. The near infrared electronic emission of singlet oxygen at 1268 nm is detected directly by novel ultrasensitive IR spectrophotometer equipment. The quantum efficiency of singlet oxygen generation by the MPO X Br- X H2O2 reaction is shown to be comparable with that of the standard chemical reaction OCl- X H2O2 at identical peroxide concentrations.     

Photogeneration of singlet molecular oxygen by the components of hematoporphyrin IX derivative

The photosensitized luminescence of singlet molecular oxygen has been studied in aqueous and alcoholic solutions of hematoporphyrin IX (HP) and di- and oligomeric components of "hematoporphyrin derivative" (photofrin II) which is known to be used as a drug in photodynamic tumor therapy. The quantum yields of 1O2 generation (gamma delta) by these compounds have been determined. It was found that the highest gamma delta values are characteristic of alcoholic and micellar detergent aqueous solutions. In detergent-free aqueous solutions containing mainly associated porphyrin molecules, gamma delta is much lower (5-30%), polymeric photofrin components being considerably less active than HP. Both localization of porphyrins in hydrophobic loci and high photosensitizing activity in lipid phase are supposed to play the key role in tumor photodestruction.     

Sensibilized dimer luminescence of singlet molecular oxygen in solutions

With the use of mechanical phosphoroscope the "universal" delayed emission has been found in aerobic solutions of different sensitizers in CCl4. The spectrum of this emission has the main maximum at 703 nm. The luminescence intensity is proportional to the square of the intensity of the exciting light. Removal of oxygen or addition of 10% of acetone led to disappearance of the luminescence. At equal intensities of singlet oxygen generation relative intensities of the 1272 and 703 nm bands differed by several orders of magnitude in solutions of different sensitizers. The energy migration from the molecules responsible for the luminescence to bacteriopheophytin and phtalocyanine has been observed. The luminescence is interpreted as dimol emission of solvated singlet molecular oxygen activated by sensitizer molecules.     

Photosensitized generation of singlet molecular oxygen by endogenous substances of the eye lens

It is shown that kinurenine derivatives, harmane (beta-carboline) and tetracycline hydrochloride known as photosensitizers of cataractogenesis in lens produce luminescence of singlet molecular oxygen (1O2) under photoexcitation in air saturated aqueous (D2O) solution. The quantum yields of the 1O2 generation by these substances are determined. The data obtained by this direct 1O2 determination technique suggest that 1O2 might take part in cataractogenesis.     

Solvent effect on the physical quenching of singlet molecular oxygen by p-quinones.

Rate constants for the interaction between singlet molecular oxygen [O2(1 delta g)] and the p-quinones 1,4-benzoquinone (BQ), duroquinone (DQ), 9,10-anthraquinone (AQ) and 1,8-dihydroxy-9,10-anthraquinone (OHAQ) are reported for several solvents at room temperature. The solvent effect on the total quenching rate constant (kt) was analysed employing the semiempirical solvatochromic equation proposed by Kamlet and Taft. The higher values of kt (2-7 x 10(7) M(-1) s(-1)) were obtained when the hydrogen-bond donor solvent ability is increased (higher alpha parameter values). The results indicate the importance of specific solvent interactions in governing the rates of the quenching.     

Interaction of singlet molecular oxygen with double fluorescent and spin sensors

Double fluorescent and spin sensors were recently used to detect transient oxidants via simultaneous fluorescence change and production of the nitroxide radical detected by electron paramagnetic resonance. One such oxidant, singlet molecular oxygen ((1)O(2)), was detected in thylakoid membrane using these probes. In the present study, we investigated the total (physical and chemical) quenching of (1)O(2) phosphorescence by sensors composed of the 2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole moiety attached to xanthene or dansyl fluorophores. We found that the quenching rate constants were in the range (2-7) x 10(7) M(-1)s(-1) in acetonitrile and D(2)O. Quenching of (1)O(2) is usually an additive process in which different functional groups may contribute. We estimated that the (1)O(2) quenching by the amine fragments was ca. one to two orders of magnitude lower than that for the complete molecules. Our data suggest that the incorporation of a fluorescent chromophore results in additional strong quenching of (1)O(2), which may in turn decrease the nitroxide yield via the (1)O(2) chemical path, possibly having an effect on quantitative interpretations. We have also found that probes with the dansyl fluorophore photosensitized (1)O(2) upon UV excitation with the quantum yield of 0.087 in acetonitrile at 366 nm. This result shows that care must be taken when the dansyl-based sensors are used in experiments requiring UV irradiation. We hope that our results will contribute to a better characterization and wider use of these novel double sensors.     

Inactivation of Neurospora crassa conidia by singlet molecular oxygen generated by a photosensitized reaction.

Photodynamic damage of Neurospora crassa conidia was studied in the presence of the photosensitizing dye, toluidine blue O. Conidia which germinated to form colonies decreased in number as irradiation time became longer. The photoinactivation of conidia was suppressed by azide, bovine serum albumin, and histidine, and was stimulated in deuterium oxide. Wild-type conidia were less sensitive to the irradiation than albino conidia. In the wild-type, carotenoid-enriched conidia were more resistant against the lethal damage than the conidia which contained small amounts of carotenoids. These results suggest that singlet molecular oxygen causes photodynamic lethal damage to N. crassa conidia and that singlet molecular oxygen is quenched by endogenous carotenoids.     

Water induced dismutation of superoxide anion generates singlet molecular oxygen

Direct spectroscopic measurement of 1268 nm singlet oxygen emission from KO2 suspensions at room temperature in three non-protonic solvents--CCl4, Cl2FCCClF2, and C6F14 by the action of water is reported. The results clearly show that the singlet oxygen generation is due to a water induced reaction, and suggest that one role of the enzyme superoxide dismutase may be the protection of biological structures, for example, lipid membranes, from degradation by singlet oxygen.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Cytochrome c enhancement of singlet molecular oxygen production by the NADPH-dependent adrenodoxin reductase-adrenodoxin system: the role of singlet oxygen in damaging adrenal mitochondrial membranes

In the presence of NADPH, cytochrome c stimulates approximately a 200-fold increase in the production of singlet oxygen by the bovine adrenodoxin reductase-adrenodoxin system. The formation of singlet oxygen, which was monitored by the attending chemiluminescence, was markedly inhibited by the addition of superoxide dismutase or 1,4-diazabicyclo[2.2.2]octane. The adrenal system, in the presence of cytochrome c, peroxidized adrenal mitochondrial lipids, as indicated by the formation of malondialdehyde. This oxidation is also inhibited by the addition of dismutase and 1,4-diazabicyclo[2.2.2]octane.     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin