Generation of reactive oxygen species in water under exposure of visible or infrared irradiation at absorption band of molecular oxygen

It is found that in bidistilled water saturated with oxygen hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sourses, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

Generation of reactive oxygen species in water under exposure to visible or infrared irradiation at absorption bands of molecular oxygen

It is found that in bidistilled water saturated with oxygen, hydrogen peroxide and hydroxyl radicals are formed under the influence of visible and infrared radiation in the absorption bands of molecular oxygen. Formation of reactive oxygen species (ROS) occurs under the influence of both solar and artificial light sources, including the coherent laser irradiation. The oxygen effect, i.e. the impact of dissolved oxygen concentration on production of hydrogen peroxide induced by light, is detected. It is shown that the visible and infrared radiation in the absorption bands of molecular oxygen leads to the formation of 8-oxoguanine in DNA in vitro. Physicochemical mechanisms of ROS formation in water when exposed to visible and infrared light are studied, and the involvement of singlet oxygen and superoxide anion radicals in this process is shown.     

叶绿体光合代谢中活性氧的产生与清除

有氧代谢不可避免产生活性氧(ROS),叶绿体的PSI和PSII反应中心均是ROS产生的主要位点。叶绿体产生的ROS主要有超氧阴离子(O2-)、过氧化氢(H2O2)、羟自由基(.OH)和单线氧(1O2),其中在PSI产生的O2-将进一步产生H2O2和.OH,而1O2产生在PSII。正常生理代谢条件下,叶绿体内抗氧化系统和光能吸收利用的调节保持活性氧产生和消灭的平衡,不会影响植物的正常生理功能。     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Mechanistic investigations of the novel non-heme vanadium bromoperoxidases. Evidence for singlet oxygen production

Three newly discovered non-heme bromoperoxidases isolated from marine algae were found to catalyze the production of singlet oxygen in reactions composed of the bromoperoxidase, hydrogen peroxide, and bromide. The bromoperoxidases studied were vanadium bromoperoxidase (V-BrPO) from Ascophyllum nodosum, native non-heme bromoperoxidase from Corallina vancouveriensis (which contains vanadium and iron), and the vanadium-reconstituted bromoperoxidase derivative from C. vancouveriensis. These enzyme systems generated near infrared emission, characteristic of singlet oxygen. The emission had a peak intensity near 1268 nm, was greatly increased in 2H2O-containing buffers, and was greatly decreased by the singlet oxygen quenchers, histidine and azide. The yield of singlet oxygen was approximately 80% of the theoretical yield. A unique feature of the non-heme bromoperoxidases distinct from the iron heme haloperoxidases, was the remarkable stability of the non-heme enzymes in the presence of singlet oxygen and oxidized bromine species. V-BrPO turned over multiple aliquots of 2 mM hydrogen peroxide without losing efficiency. In contrast, iron heme lactoperoxidase was completely inactivated after turnover of the first aliquot of 2 mM hydrogen peroxide, and iron heme chloroperoxidase was 50% deactivated. The profile of singlet oxygen formation by V-BrPO and the near stoichiometric yield of singlet oxygen suggest that the mechanism of singlet oxygen formation is the same as the mechanism of dioxygen formation determined by oxygen probe measurements.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Light and oxygen are not required for harpin-induced cell death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.     

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.     

Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as β-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease.     

DNA fragmentation of human lymphocytes in dynamics of development of apoptosis induced by action of UV radiation and reactive oxygen species

It was established that UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m²; reactive oxygen species (ROS); and singlet oxygen induce the DNA fragmentation of human lymphocytic cells 20 h after exposure. Using DNA comet assay, DNA damage (monostrand breaks) has been revealed to occur immediately after the UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² or after the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/l (type-C1 comets) and to reach a maximum 6 h after action on cells of UV light or ROS (type-C2 and -C3 comets). A suggestion has been made about the leading role of the p53-dependent pathway in apoptosis in human lymphocytes under the conditions of the action of UV light and ROS.