Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

The decomposition of peroxynitrite does not yield nitroxyl anion and singlet oxygen

In a recent article [Khan, A. U., Kovacic, D., Kolbanovsky, A., Desai, M., Frenkel, K. & Geacintov, N. E. (2000) Proc. Natl. Acad. Sci. USA 97, 2984–2989], the authors claimed that ONOO−, after protonation to ONOOH, decomposes into 1HNO and 1O2 according to a spin-conserved unimolecular mechanism. This claim was based partially on their observation that nitrosylhemoglobin is formed via the reaction of peroxynitrite with methemoglobin at neutral pH. However, thermochemical considerations show that the yields of 1O2 and 1HNO are about 23 orders of magnitude lower than those of ⋅NO2 and ⋅OH, which are formed via the homolysis of ONOOH. We also show that methemoglobin does not form with peroxynitrite any spectrally detectable product, but with contaminations of nitrite and H2O2 present in the peroxynitrite sample. Thus, there is no need to modify the present view of the mechanism of ONOOH decomposition, according to which initial homolysis into a radical pair, [ONO⋅ ⋅OH]cage, is followed by the diffusion of about 30% of the radicals out of the cage, while the rest recombines to nitric acid in the solvent cage.

Peroxynitrite does not decompose to singlet oxygen (1ΔgO2) and nitroxyl (NO−)

According to Khan et al. [Khan, A. U., Kovacic, D., Kolbanovskiy, A., Desai, M., Frenkel, K. & Geacintov, N. E. (2000) Proc. Natl. Acad. Sci. USA 97, 2984–2989], peroxynitrite (ONOO−) decomposes after protonation to singlet oxygen (1ΔgO2) and singlet oxonitrate (nitroxyl, 1NO−) in high yield. They claimed to have observed nitrosyl hemoglobin from the reaction of NO− with methemoglobin; however, contamination with hydrogen peroxide gave rise to ferryl hemoglobin, the spectrum of which was mistakenly assigned to nitrosyl hemoglobin. We have carried out UV–visible and EPR experiments with methemoglobin and hydrogen peroxide-free peroxynitrite and find that no NO− is formed. With this peroxynitrite preparation, no light emission from singlet oxygen at 1270 nm is observed, nor is singlet oxygen chemically trapped; however, singlet oxygen was trapped when hydrogen peroxide was also present, as previously described [Di Mascio, P., Bechara, E. J. H., Medeiros, M. H. G., Briviba, K. & Sies, H. (1994) FEBS Lett. 355, 287–289]. Quantum mechanical and thermodynamic calculations show that formation of the postulated intermediate, a cyclic form of peroxynitrous acid (trioxazetidine), and the products 1NO− and 1ΔgO2 requires Gibbs energies of ca. +415 kJ⋅mol−1 and ca. +180 kJ⋅mol−1, respectively. Our results show that the results of Khan et al. are best explained by interference from contaminating hydrogen peroxide left from the synthesis of peroxynitrite.

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: Comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1-dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4-triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen $\rm(\sp1O\sb2).$ Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly $\sp{\prime\prime}2+2\sp{\prime\prime}$ adducts and a small amount of the "ene" adducts. The $\sp{\prime\prime}2+2\sp{\prime\prime}$ is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD. ^

Reactions of singlet oxygen and singlet oxygen mimics with dicyclopropylethylenes: comparison of reactivity and mechanisms

An extensive study of the reaction pathways of 1,1- dicyclopropyl ethylene, cis- and trans- 1,2-dicyclopropylethylenes has been undertaken with different electrophiles 4-methyl-1,2,4- triazoline-3,5-dione (MTAD), tetracyanoethylene (TCNE), and singlet oxygen (102). Comparison of reactivity and reaction mechanisms among the electrophiles is investigated. Singlet oxygen exhibits significantly lower reactivity compared to the other electrophiles. MTAD and TCNE react with dicyclopropylethylenes to produce predominantly "2+2" adducts and a small amount of the "ene" adducts. The "2+2" is the major product presumably because of the high activation energy leading to the highly strained "ene" products. Solvent trapping studies provide strong evidence of a "stepwise" mechanism, involving a zwitterionic or aziridinium imide as an intermediate from the study of the reactions products of dicyclopropylethylenes and MTAD.

Oxidations of thio ketones by singlet and triplet oxygen

Oxidation of di-tert-butyl thioketone (1) and 2,2,4,4-tetramethylcyclobutylth ioketone (2) by singlet oxygen yields the corresponding sulfine and ketone; in the case of 1 the sulfine is the major product, whereas in 2 it is the ketone. 1,2,3-Dioxathietane has been suggested as the precursor for the ketones, and the zwitterionic/diradid peroxide is believed to be a common primary intermediate for both sulfine and ketone. Steric influence is felt both during primary interaction between singlet oxygen and thioketone and during the partitioning of the peroxide intermediate. Steric interaction is suggested as the reason for variations in the product distribution between 1 and 2. Singlet oxygen is also generated through energy transfer from the triplet state of thioketones. These excited states also directly react with oxygen to yield ketone.

Oxidations of thio ketones by singlet and triplet oxygen

Oxidation of di-tert-butyl thioketone (1) and 2,2,4,4-tetramethylcyclobutylth ioketone (2) by singlet oxygen yields the corresponding sulfine and ketone; in the case of 1 the sulfine is the major product, whereas in 2 it is the ketone. 1,2,3-Dioxathietane has been suggested as the precursor for the ketones, and the zwitterionic/diradid peroxide is believed to be a common primary intermediate for both sulfine and ketone. Steric influence is felt both during primary interaction between singlet oxygen and thioketone and during the partitioning of the peroxide intermediate. Steric interaction is suggested as the reason for variations in the product distribution between 1 and 2. Singlet oxygen is also generated through energy transfer from the triplet state of thioketones. These excited states also directly react with oxygen to yield ketone.

Cytochrome c-promoted cardiolipin oxidation generates singlet molecular oxygen

The interaction of cytochrome c (cyt c) with cardiolipin (CL) induces protein conformational changes that favor peroxidase activity. This process has been correlated with CL oxidation and the induction of cell death. Here we report evidence demonstrating the generation of singlet molecular oxygen [O-2((1)Delta(g))] by a cyt c-CL complex in a model membrane containing CL. The formation of singlet oxygen was directly evidenced by luminescence measurements at 1270 nm and by chemical trapping experiments. Singlet oxygen generation required cyt c-CL binding and occurred at pH values higher than 6, consistent with lipid-protein interactions involving fully deprotonated CL species and positively charged residues in the protein. Moreover, singlet oxygen formation was specifically observed for tetralinoleoyl CL species and was not observed with monounsaturated and saturated CL species. Our results show that there are at least two mechanisms leading to singlet oxygen formation: one with fast kinetics involving the generation of singlet oxygen directly from CL hydroperoxide decomposition and the other involving CL oxidation. The contribution of the first mechanism was clearly evidenced by the detection of labeled singlet oxygen [O-18(2)((1)Delta(g))] from liposomes supplemented with 18-oxygen-labeled CL hydroperoxides. However quantitative analysis showed that singlet oxygen yield from CL hydroperoxides was minor (<5%) and that most of the singlet oxygen is formed from the second mechanism. Based on these data and previous findings we propose a mechanism of singlet oxygen generation through reactions involving peroxyl radicals (Russell mechanism) and excited triplet carbonyl intermediates (energy transfer mechanism).

Electron Spectroscopic Studies of Oxygen and Carbon Dioxide Adsorbed on Metal Surfaces

Adsorption of oxygen on Ni, Cu, Pd, Ag, and Au surfaces has been investigated by employing UV and X-ray photoelectron spectrscopy as well as electron energy loss spectroscopy (EELS). Molecularly chemisorbed (singlet) oxygen is found on Ni, Cu, Ag, and Au surfaces showing features such as stabilization of the rB* orbital, destabilization of the .nu orbital, higher O(1s) binding energy than the atomic species, and a band 2-3 eV below the Fermi level due to metal d-O(2p)u* interaction. 0-0 and metal-oxygen stretching frequencies have been observed in EELS. Physical adsorption of O2 is found to occur on Pd and Ni surfaces, only at high exposures in the latter case. Physical adsorption and multilayer condensation of CO, on metal surfaces are distinguished by characteristic relaxation shifts in UPS as well as O(1s) binding energies. Adsorption of CO on a Ni surface covered with presorbed atomic oxygen gives rise to C02.

Stress Resistance and Disease Resistance in Seaweeds:The Role of Reactive Oxygen Metabolism

It has become clear that the last 15-20 years that the immediate effect of a wide range of environmental stresses,and of infection,on vascular plants is to increase the information of reactive oxygen species(ROS) and to impose oxidative stress on the cells.Since 1994,sufficient examples similar responses in a broad range of marine macroalgae have been decribed to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond(and become resistant) to stress and infection.Desiccation,freezing,low temperatures,high light,ultraviolet radiation,and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen.The response to other stresses (infection or oligosaccharides which signal that infection is occurring,mechanical stress,hyperosmotic shock) is quite different-a more rapid and intence,but short-lived production of ROS ,discribed as an "oxidative burst"-which is attributed to activation of NADPHoxidases in the plasma membrane.Seaweed species that are able to survive such stresses or resist infection have the capacity to remove the ROS through a high cellular content of antioxidant compounds,or a high activity of antioxidant enzymes.

Stress resistance and disease resistance in seaweeds: The role of reactive oxygen metabolism

It has become clear over the last 15-20 years that the immediate effect of a wide range of environmental stresses, and of infection, on vascular plants is to increase the formation of reactive oxygen species (ROS) and to impose oxidative stress on the cells. Since 1994, sufficient examples of similar responses in a broad range of marine macroalgae have been described to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond (and become resistant) to stress and infection. Desiccation, freezing, low temperatures, high light, ultraviolet radiation, and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen. The response to other stresses (infection or oligosaccharides which signal that infection is occurring, mechanical stress, hyperosmotic shock) is quite different-a more rapid and intense, but short-lived production of ROS, described as an

Phototransformation of hematoporphyrin in aqueous solution: Anomalous behavior at low oxygen concentration

The photoactivation of a photosensitizer is the initial step in photodynamic therapy (PDT) where photochemical reactions result in the production of reactive oxygen species and eventually cell death. In addition to oxidizing biomolecules, some of these photochemical reactions lead to photosensitizer degradation at a rate dependent on the oxygen concentration among other factors. We investigated photodegradation of Photogem A (R) (28 mu M), a hematoporphyrin derivative, at different oxygen concentrations (9.4 to 625.0 mu M) in aqueous solution. The degradation was monitored by fluorescence spectroscopy. The degradation rate (M/s) increases as the oxygen concentration increases when the molar ratio of oxygen to PhotogemA (R) is greater than 1. At lower oxygen concentrations (< 25 mu M) an inversion of this behavior was observed. The data do not fit a simple kinetic model of first-order dependence on oxygen concentration. This inversion of the degradation rate at low oxygen concentration has not previously been demonstrated and highlights the relationship between photosensitizer and oxygen concentrations in determining the photobleaching mechanism(s). The findings demonstrate that current models for photobleaching are insufficient to explain completely the effects at low oxygen concentration.

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.

Synthesis, crystal structures, DNA binding and cleavage activity of L-glutamine copper(II) complexes of heterocyclic bases

Ternary L-glutamine (L-gln) copper(II) complexes [Cu(L-gln)(B)(H2O)](X) (B = 2,2'-bipyridine (bpy), X = 0.5SO(4)(2-), 1; B = 1,10-phenanthroline (phen), X = ClO4-, 2) and [Cu(L-gln)(dpq)(ClO4)] (3) (dpq, dipyridoquinoxaline) are prepared and characterized by physicochemical methods. The DNA binding and cleavage activity of the complexes have been studied. Complexes 1-3 are structurally characterized by X-ray crystallography. The complexes show distorted square pyramidal (4+1) CuN3O2 coordination geometry in which the N,O-donor amino acid and the N, N-donor heterocyclic base bind at the basal plane with a H2O or perchlorate as the axial ligand. The crystal structures of the complexes exhibit chemically significant hydrogen bonding interactions besides showing coordination polymer formation. The complexes display a d-d electronic band in the range of 610-630 nm in aqueous-dimethylformamide (DMF) solution (9:1 v/v). The quasireversible cyclic voltammetric response observed near -0.1 V versus SCE in DMF-TBAP is assignable to the Cu(II)/Cu(I) couple. The binding affinity of the complexes to calf thymus (CT) DNA follows the order: 3 (dpq) > 2 (phen) >> 1 (bpy). Complexes 2 and 3 show DNA cleavage activity in dark in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent via a mechanistic pathway forming hydroxyl radical as the reactive species. The dpq complex 3 shows efficient photoinduced DNA cleavage activity on irradiation with a monochromatic UV light of 365 nm in absence of any external reagent. The cleavage efficiency of the DNA minor groove binding complexes follows the order:3 > 2 >> 1. The dpq complex exhibits photocleavage of DNA on irradiation with visible light of 647.1 nm. Mechanistic data on the photo-induced DNA cleavage reactions reveal the involvement of singlet oxygen (O-1(2)) as the reactive species in a type-II pathway. (C) 2008 Elsevier B.V. All rights reserved.