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).     

On the antioxidant properties of therapeutic drugs: quenching of singlet molecular oxygen by aminosalicylic acids

The ability of the widely employed therapeutic drugs 4-aminosalicylic acid and 5-aminosalicylic acid to act as singlet molecular oxygen (O(2)((1)delta(g))) scavengers was investigated at pH 7 and pH 12. The isomer 3-aminosalicylic acid was also included in the study for comparative purposes. All three compounds quench photochemically generated O(2)((1)delta(g)) with rate constants in the range of 10(7)-10(8) x M(-1)s(-1), depending on the experimental conditions. No chemical reaction (oxidation of the aminosalicylic acids) was detected at the neutral pH, whereas at pH 12 both chemical and physical interactions with O(2)((1)delta(g)) operated. The physical process implies the de-activation of the oxidant species without destruction of the aminosalicylic acid. The quotients between the overall and reactive rate constants for O(2)((1)delta(g)) quenching at pH 12 (k(r)/k(t) ratios), which account for the actual effectiveness of photodegradation, were relatively low (0.22, 0.04, and 0.06 for 3-, 4- and 5-aminosalicylic acids, respectively). This indicates that the drugs, particularly the 4- and 5-amino derivatives, de-activate the excited oxygen species, at both pH values studied, mainly in a physical fashion, preventing its photodegradation and providing an antioxidative protection for possible photo-oxidizable biological targets in the surroundings.     

Chemical quenching of singlet oxygen by carotenoids in plants

Carotenoids are considered to be the first line of defense of plants against singlet oxygen ((1)O(2)) toxicity because of their capacity to quench (1)O(2) as well as triplet chlorophylls through a physical mechanism involving transfer of excitation energy followed by thermal deactivation. Here, we show that leaf carotenoids are also able to quench (1)O(2) by a chemical mechanism involving their oxidation. In vitro oxidation of β-carotene, lutein, and zeaxanthin by (1)O(2) generated various aldehydes and endoperoxides. A search for those molecules in Arabidopsis (Arabidopsis thaliana) leaves revealed the presence of (1)O(2)-specific endoperoxides in low-light-grown plants, indicating chronic oxidation of carotenoids by (1)O(2). β-Carotene endoperoxide, but not xanthophyll endoperoxide, rapidly accumulated during high-light stress, and this accumulation was correlated with the extent of photosystem (PS) II photoinhibition and the expression of various (1)O(2) marker genes. The selective accumulation of β-carotene endoperoxide points at the PSII reaction centers, rather than the PSII chlorophyll antennae, as a major site of (1)O(2) accumulation in plants under high-light stress. β-Carotene endoperoxide was found to have a relatively fast turnover, decaying in the dark with a half time of about 6 h. This carotenoid metabolite provides an early index of (1)O(2) production in leaves, the occurrence of which precedes the accumulation of fatty acid oxidation products.     

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     

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.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ leads to plastoquinone‐9 (PQ‐9), which is subsequently oxidized to hydroxyplastoquinone‐9 [PQ(OH)‐9]. We provide here evidence that oxidation of PQ(OH)‐9 by ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.     

Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with catechins (catechin (CA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG)) and related compounds (5-methoxyresorcinol (MR), 4-methylcatechol (MC), and n-propyl gallate (PG)) was performed in ethanol at 35 degrees C. MR, MC, and PG are considered to be a model of resorcinol (A)-, catechol (B)-, and gallate (G)-rings in catechins, respectively. The overall rate constants, k(Q) (= k(q) + k(r), physical quenching + chemical reaction), for the reaction of catechins with (1)O(2) increased in the order of PG < MR < MC < CA < EC < EGC < ECG < EGCG. In a comparison of the rate constants, the relationship between quenching rates and chemical structures is discussed. The catechins which have lower peak oxidation potentials, E(P), show higher reactivities. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by catechins. The k(Q) values of EGCG (1.47 x 10(8) M(-1) s(-1)) and ECG (7.81 x 10(7)) were found to be larger than those of lipids (1.3 x 10(5)-1.9 x 10(5) M(-1) s(-1)), amino acids (<3.7 x 10(7)), and DNA (5.1 x 10(5)). Further, these values are similar to those (1.15 x 10(8)-2.06 x 10(8) M(-1) s(-1)) of alpha- and gamma-tocopherol, ubiquinol-10, and gamma-tocopherol hydroquinone (plastoquinol model). The result suggests that catechins may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Physical and chemical scavenging of singlet molecular oxygen by tocopherols

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers-determination of singlet oxygen quenching rate constants and oxidation products

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.     

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.