Vitamin B-sensitized photo-oxidation of dopamine

Abstract Kinetics and mechanism of the photo-oxidation of the natural catecholamine-type neurotransmitter dopamine (DA) has been studied in aqueous solution, under aerobic conditions, in the presence of riboflavin (Rf, vitamin B(2)) as a photosensitizer. Results indicate the formation of a weak dark complex Rf-DA, with a mean apparent association constant K(ass) = 30 m(-1), only detectable at DA concentrations much higher than those employed in photochemical experiments. An intricate mechanism of competitive reactions operates upon photoirradiation. DA quenches excited singlet and triplet states of Rf, with rate constants of 4.2 x 10(9) and 2.2 x 10(9) m(-1) s(-1), respectively. With the catecholamine in a concentration similar to that of dissolved molecular oxygen in air-saturated water, DA and oxygen competitively quench the triplet excited state of Rf, generating superoxide radical anion (O(2)(*-)) and singlet molecular oxygen (O(2)((1)Delta(g))) by processes initiated by electron and energy-transfer mechanisms, respectively. Rate constants values of 1.9 x 10(8) and 6.6 x 10(6) m(-1) s(-1) have been obtained for the overall and reactive (chemical) interaction of DA with O(2)((1)Delta(g)). The presence of superoxide dismutase increases both the observed rates of aerobic DA photo-oxidation and oxygen uptake, due to its known catalytic scavenging of O(2)(*-), a species that could revert the overall photo-oxidation effect, according to the proposed reaction mechanism. As in most of the catecholamine oxidative processes described in the literature, aminochrome is the DA oxidation product upon visible light irradiation in the presence of Rf. It is generated with a quantum yield of 0.05.     

Singlet oxygen generated from the decomposition of peroxymonocarbonate and its observation with chemiluminescence method

The decomposition of peroxymonocarbonate (HCO(4)(-)) has been investigated by flow-injection chemiluminescence (CL) method. An ultraweak CL was observed during mixing the bicarbonate and hydrogen peroxide solution in organic cosolvent. An appropriate amount of fluorescent organic compounds, such as dichlorofluorescein (DCF), was added to the HCO(4)(-) solution, a strong CL was recorded. Based on studies of the spectrum of fluorescence, CL and UV-vis spectra, electron spin trapping (ESR) technique, mass spectra (MS) and comparison with H(2)O(2)/hypochlorite (ClO(-)) and H(2)O(2)/molybdate (MoO(4)(-)) systems, the CL mechanism was proposed. The reaction is initiated by unimolecular homolysis of the peroxo O-O bond in HO-OCOO(-) molecule. It was suggested that the bond rearrangement within radicals yield superoxide ion (O(2)(*-)). The interaction of superoxide ion with perhydroxyl radical produces singlet oxygen ((1)O(2)). The energy transfers from singlet oxygen to DCF forming an excited energy acceptor (DCF*). Luminescence (lambda(max)=509 nm) was emitted during the relaxation of the energy acceptor to the ground state.     

Oxidation of di- and tripeptides of tyrosine and valine mediated by singlet molecular oxygen, phosphate radicals and sulfate radicals.

Kinetics and mechanism of the oxidation of tyrosine (Tyr) and valine (Val) di- and tripeptides (Tyr-Val, Val-Tyr and Val-Tyr-Val) mediated by singlet molecular oxygen [O(2)((1)Delta(g))], phosphate (HPO(4)(*-) and PO(4)(*2-)) and sulfate (SO(4)(*-)) radicals was studied, employing time-resolved O(2)((1)Delta(g)) phosphorescence detection, polarographic determination of dissolved oxygen and flash photolysis. All the substrates were highly photooxidizable through a O(2)((1)Delta(g))-mediated mechanism. Calculated quotients between the overall and reactive rate constants for the quenching of O(2)((1)Delta(g)) by Tyr-derivatives (k(t)/k(r) values, accounting for the efficiency of the effective photooxidation) were 1.3 for Tyr, 1 for Tyr-Val, 2.8 for Val-Tyr and 1.5 for Val-Tyr-Val. The effect of pH on the kinetics of the photooxidative process confirms that the presence of the dissociated phenolate group of Tyr clearly dominates the O(2)((1)Delta(g)) quenching process. Products analysis by LC-MS indicates that the photooxidation of Tyr di- and tripeptides proceeds with the breakage of peptide bonds. The information obtained from the evolution of primary amino groups upon photosensitized irradiation is in concordance with these results. Absolute rate constants for the reactions of phosphate radicals (HPO(4)(*-) and PO(4)(*2-), generated by photolysis of the P(2)O(8)(4-) at different pH) and sulfate radicals (SO(4)(*-), produced by photolysis of the S(2)O(8)(2-)) with Tyr peptides indicate that for all the substrates, the observed tendency in the rate constants is: SO(4)(*-) > or = HPO(4)(*-) > or = PO(4)(*2-). Formation of the phenoxyl radical of tyrosine was detected as an intermediate involved in the oxidation of tyrosine by HPO(4)(*-).     

Superoxide dismutase versus ferricytochrome C: determining rate constants for the spin trapping of superoxide by cyclic nitrones

Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.     

Photo-induced reactive oxygen species generation by different water-soluble fullerenes (C) and their cytotoxicity in human keratinocytes

Abstract In this study we report the phototoxicity toward HaCaT keratinocytes that results from the photogeneration of superoxide and singlet oxygen ((1)O(2)) by four different "water-soluble" fullerene (C(60)) preparations-monomeric (gamma-CyD)(2)/C(60) (gamma-cyclodextrin bicapped C(60)) and three aggregated forms-THF/nC(60) (prepared by solvent exchange from THF solution); Son/nC(60) (prepared by sonication of a toluene/water mixture); and gamma-CyD/nC(60) (prepared by heating the [gamma-CyD](2)/C(60) aqueous solution). Our results demonstrate that all four C(60) preparations photogenerate (1)O(2) efficiently. However, the properties of C(60)-generated (1)O(2), including its availability for reactions in solution, are markedly different for the monomeric and aggregated forms. (1)O(2) produced by monomeric (gamma-CyD)(2)/C(60) is quenchable by NaN(3) and its quantum yield in D(2)O, which is only weakly dependent on oxygen concentration, is as high as C(60) in toluene. In contrast, (1)O(2) generated from aggregated C(60) is not quenchable by NaN(3), exhibits a solvent-independent short-lived lifetime (ca 2.9 mus), is highly sensitive to oxygen concentration while its phosphorescence is redshifted. All these features indicate that (1)O(2) is sequestered inside the C(60) aggregates, which may explain why these preparations were not phototoxic toward HaCaT cells. Electron paramagnetic resonance studies demonstrated the generation of the C(60) anion radical (C(60)(*-)) when (gamma-CyD)(2)/C(60) was irradiated (lambda > 300 nm) in the presence of a reducing agent (NADH); spin trapping experiments (lambda > 400 nm) with 5,5-dimethyl-1-pyrroline N-oxide clearly showed the generation of superoxide resulting from the reaction of C(60)(*-) with oxygen. In vitro tests with HaCaT keratinocytes provided evidence that (gamma-CyD)(2)/C(60) phototoxicity is mainly mediated by (1)O(2) (Type II mechanism) with only a minor contribution from free radicals (Type I mechanism).     

DNA cleavage by UVA irradiation of NADH with dioxygen via radical chain processes

Efficient DNA cleaving-activity is observed by UVA irradiation of an O(2)-saturated aqueous solution of NADH (beta-nicotinamide adenine dinucleotide, reduced form). No DNA cleavage has been observed without NADH under otherwise the same experimental conditions. In the presence of NADH, energy transfer from the triplet excited state of NADH ((3)NADH*) to O(2) occurs to produce singlet oxygen ((1)O(2)) that is detected by the phosphorescence emission at 1270 nm. No quenching of (1)O(2) by NADH was observed as indicated by no change in the intensity of phosphorescence emission of (1)O(2) at 1270 nm in the presence of various concentrations of NADH. In addition to the energy transfer, photoinduced electron transfer from (3)NADH* to O(2) occurs to produce NADH(*+) and O(2)(*-), both of which was observed by ESR. The quantum yield of the photochemical oxidation of NADH with O(2) increases linearly with increasing concentration of NADH but decreases with increasing the light intensity absorbed by NADH. Such unusual dependence of the quantum yield on concentration of NADH and the light intensity absorbed by NADH indicates that the photochemical oxidation of NADH with O(2) proceeds via radical chain processes. The O(2)(*-) produced in the photoinduced electron transfer is in the protonation equilibrium with HO(2)(*), which acts as a chain carrier for the radical chain oxidation of NADH with O(2) to produce NAD(+) and H(2)O(2), leading to the DNA cleavage.     

Antioxidant and anticancer activity of fresh corm extract from Romulea tempskyana (Iridaceae)

The antioxidant and anticancer properties of a medicinal plant, Romulea tempskyana (R. tempskyana) (Iridaceae) were investigated. The fresh corm water extract of R. tempskyana significantly increased cell viability against H(2)O(2) cytotoxicity(.) The extract also showed high inhibition of the lipid peroxidation activity (78.20%), reducing power (IC(50) 64.99?μg?mL(-1)) activity and hydroxyl (IC(50) 38.66?μg?mL(-1)), superoxide (IC(50) 25.99?μg?ml(-1)) and DPPH (IC(50) 19.88?μg?mL(-1)) radical scavenging activity. On the other hand, treatment of the cells with higher extract concentration showed the anticancer activity inducing cytotoxicity. The extract significantly affected Hepatoma G2 and H1299 cell proliferation (IC(50); 103.79 and 88.15?μg?mL(-1)). The amount of MDA (2-fold and 2.5-fold) and activities of several cellular antioxidant enzymes, including Se-GSPx (30%, 15%), non-Se-GSH-Px (11%, 16%) and GST (17%, 23%) increased in Hepatoma G2 and H1299 cells treated with IC(50) concentrations of extract, respectively. These findings suggest that R. tempskyana extract exhibits antioxidant and carcinogenesis-reducing potential.     

Antioxidative properties of probucol estimated by the reactivity with superoxide and by electrochemical oxidation

The reaction of probucol with superoxide (O2(*-)) was investigated in acetonitrile using both electron spin resonance (ESR) and electrochemical techniques. The formation of phenoxyl radical was observed during the reaction of probucol with O2(*-) by ESR spectroscopy. The reaction of probucol with O2(*-) in acetonitrile was followed by cyclic voltammetry. With the addition of probucol, the oxidation peak current of O2(*-) decreased concentration dependently. This suggests that probucol reacts with O2(*-), that is, probucol scavenges O2(*-) in acetonitrile. 2,6-Di-tert-butyl-p-benzoquinone was identified as the major product of the reaction of probucol with O2(*-) in acetonitrile. Electrochemical oxidation of probucol was also performed. Probucol gives an irreversible oxidation peak at ca. +1.4 V vs. the saturated calomel electrode in the cyclic voltammogram. Controlled-potential electrolysis was carried out at +1.2 V in a divided cell. 2,6-Di-tert-butyl-p-benzoquinone, 4,4'-dithiobis(2,6-di-tert-butylphenol), and 4,4'-trithiobis(2,6-di-tert-butylphenol) were identified as the products of anodic oxidation. These redox properties of probucol may correlate with the physiological activities.     

Mechanism of TiO2-assisted photocatalytic degradation of dyes under visible irradiation: photoelectrocatalytic study by TiO2-film electrodes

Photoelectrocatalytic degradation of various dyes under visible light irradiation with a TiO(2) nanoparticles electrode has been investigated to reveal the mechanism for TiO(2)-assisted photocatalytic degradation of dyes. The degradation of both cationic and anionic dyes at different biases, including the change in the degradation rate of the dyes and the photocurrent change with the bias potential, the degraded intermediates, the voltage-induced adsorption of dyes, the accumulation of electrons in the TiO(2) electrode, the effect of various additives such as benzoquinone (BQ) and N,N-dimethyl aniline (DMA), and the formation of active oxygen species such as O(2)(*-) and H(2)O(2) were examined by UV-visible spectroscopy, HPLC, TOC, and spin-trap ESR spectrometry. It was found that the dyes could controllably interact with the TiO(2) surface by external bias changes and charging of dyes. The cationic dyes such as RhB and MG underwent efficient mineralization at negative bias, but the N-dealkylation process predominated at positive bias under visible light irradiation. The discolorations of the anionic dyes SRB and AR could not be accelerated significantly at either negative or positive bias. At a negative bias of -0.6 V vs SCE, O(2)(*-) and dye(*+) were formed simultaneously at the electrode/electrolyte interface during degradation of cationic RhB. In the case of anionic dyes, however, it is impossible for the O(2)(*-) and dye cationic radical to coexist at the electrode/electrolyte surface. Experimental results imply both the superoxide anionic radical and the dye cationic radical are essential to the mineralization of the dyes under visible light-induced photocatalytic conditions.     

Visible photooxidation of dibenzothiophenes sensitized by 2-(4-methoxyphenyl)-4, 6-diphenylpyrylium: an electron transfer mechanism without involvement of superoxide

We report here on a new electron-transfer mechanism for visible-light photooxidation of sulfides in which no superoxide ion is involved. Visible-light irradiation of 2-(4-methoxyphenyl)-4, 6-diphenylpyrylium tetrafluoroborate (MOPDPP(+)BF(4)(-)) in an O(2)-saturated acetonitrile solution containing dibenzothiophene (DBT) results in nearly 100% conversion to oxygenated products, DBT sulfoxide and sulfone. The photooxidation of DBT is initiated by a photoinduced electron-transfer process, where the excited MOPDPP(+) traps an electron from the ground-state DBT to form MOPDPP(*) and DBT radical cation. Such a mechanism is consistent with the studies of laser flash photolysis, electron spin resonance, and fluorescence quenching of the irradiated system. The photogenerated DBT radical cation undergoes a coupling reaction with O(2) to produce the intermediate responsible for the formation of the oxygenated products. The presence of O(2) has no effect on the decay kinetics of the transient absorption of MOPDPP(*), indicating that no redox reaction occurs between MOPDPP(*) and O(2), and thus no superoxide ion (O(2)(*-)) is formed. Moreover, the ESR signal of MOPDPP(*) was significantly enhanced in the presence of O(2), consistent with the assumption that the photogenerated DBT radical cation couples with O(2) to form the oxygen-adduct, which is subject to further reactions (Scheme 3) leading to the final oxygenated products. Similar results have been obtained when using 10-methylacridine hexafluorophosphate (AcrH(+)PF(6)(-), which has similar reduction potential in the ground state as MOPDPP(+)) as the sensitizer. This finding provides a possibility for the photooxidation of sulfides with dioxygen utilizing visible light (solar energy) and is also of significance in clarification of the reaction mechanism.     

Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2-* versus 1O2

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.     

Singlet oxygen reacts with 2',7'-dichlorodihydrofluorescein and contributes to the formation of 2',7'-dichlorofluorescein

Abstract There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(*2+)) and semi-reduced (MB(*)) radicals of the sensitizer and subsequently super-oxide radical (O(2)(*-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(*-), indicating that both (1)O(2) and MB(*2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(*2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(*2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).     

Protective effect of Castanea sativa and Quercus robur leaf extracts against oxygen and nitrogen reactive species

Topical natural antioxidants are a useful strategy for the prevention of photoaging and oxidative stress mediated skin diseases. In view of this underlying principle, the screening of natural plant extracts with scavenging activity for pro-oxidant reactive species is a primary requirement for the development of new topical antioxidant formulations. In the present study, an ethanol:water (7:3) extract from Castanea sativa leaves and a ethanol:water (2:3) extract from Quercus robur leaves were evaluated for their putative in vitro scavenging effects on reactive oxygen species (ROS) namely superoxide radical (O(2)(-)), hydroxyl radical (HO()), peroxyl radical (ROO()), hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)) as well as on reactive nitrogen species (RNS) namely nitric oxide (()NO) and peroxynitrite (ONOO(-)). The extracts presented a high potency to scavenge the tested reactive species, all the IC(50)s being found at the microg/mL level. IC(50)s (mean+/-SE) for the ROS O(2)(-),HO(),H(2)O(2) and (1)O(2) were 13.6+/-1.8; 216+/-4; 410+/-8; 12.3+/-0.7 microug/mL, respectively, for C. sativa, and 11.0+/-0.5; 285+/-22; 251+/-32; 7.90+/-0.56 microg/mL, respectively, for Q. robur. The ORAC values obtained for ROO() were 1.24+/-0.13 for C. sativa and 1.09+/-0.06 for Q. robur. The IC(50)s (mean+/-SE) for ()NO and ONOO(-) were 3.10+/-0.14 and 1.49+/-0.10 microg/mL, respectively, for C. sativa and 3.13+/-0.11 and 0.95+/-0.02 microg/mL, respectively, for Q. robur. The content of total phenolics for C. sativa and Q. robur were 284+/-9 and 346+/-4 mg of gallic acid equivalents (GAE)/g of lyophilized extract respectively. The observed effects might be of relevance considering the putative interest of these extracts as topical antioxidants.     

Antioxidant activity and superoxide anion radical interaction with 2-(ferrocenylmethylamino) benzonitrile and 3-(ferrocenylmethylamino) benzonitrile

Cyclic voltammetry was used to explore the interaction of (ferrocenylmethylamino) benzonitrile (FMAB) with superoxide anion radical (\({\text{O}}_{2}^{. - }\)), electrochemically generated by the reduction in commercial molecular oxygen in acetonitrile. The difference in the electrochemical behavior of \({\text{O}}_{2}^{. - }\) in the absence and presence of FMAB, including shifts in peak potential and decrease in anodic peak current, was successfully investigated for the determination of interaction parameters such as the binding constant, ratio of binding constants, binding free energy and mode of interaction. The anodic peak potential shifts and the magnitude of binding free energy ΔG suggest the electrostatic interaction of \({\text{O}}_{2}^{. - }\) with FMAB as the dominant mode, whereas the negative sign of ΔG indicates the spontaneity of the interaction. The antioxidant activity of FMAB derivatives was also evaluated using spectrophotometrical and electrochemical techniques. The spectrophotometrical assays were carried out using 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), while the electrochemical assays were determined by measuring the oxidation peak current of \({\text{O}}_{2}^{. - }\). The activity was found to be highest for 3FMAB in both DPPH and \({\text{O}}_{2}^{. - }\) radicals scavenging methods (0.0183) and (0.0090 mg/mL), respectively, which is equal to half the antioxidant activity of standard antioxidant ascorbic acid (0.0117) and (0.0041 mg/mL).     

Kinetic analysis of superoxide anion radical-scavenging and hydroxyl radical-scavenging activities of platinum nanoparticles

There are few reports on the physiological effects of metal nanoparticles (nps), especially with respect to their functions as scavengers for superoxide anion radical (O2(.-)) and hydroxyl radical (.OH). We tried to detect the scavenging activity of Pt nps using a hypoxanthine-xanthine oxidase system for O2(.-) and using a Fenton and a UV/H2O2 system for .OH. Electron spin resonance analysis revealed that 2 nm particle size Pt nps have the ability to scavenge O2(.-) and .OH. The calculated rate constant for the O2(.-)-scavenging reaction was 5.03 +/- 0.03 x 10(7) M (-1) s (-1). However, the analysis of the Fenton and UV/H 2O 2 system in the presence of Pt nps suggested that the .OH-scavenging reaction cannot be determined in both systems. Among particle sizes tested from 1 to 5 nm, 1 nm Pt nps showed the highest O2(.-)-scavenging ability. Almost no cytotoxicity was observed even after adherent cells (TIG-1, HeLa, HepG2, WI-38, and MRC-5) were exposed to Pt nps at concentrations as high as 50 mg/L. Pt nps scavenged intrinsically generated reactive oxygen species (ROS) in HeLa cells. Additionally, Pt nps significantly reduced the levels of intracellular O2(.-) generated by UVA irradiation and subsequently protected HeLa cells from ROS damage-induced cell death. These findings suggest that Pt nps may be a new type of antioxidant capable of circumventing the paradoxical effects of conventional antioxidants.     

Direct observation of the one-electron reduction of methyl viologen mediated by the CO2 radical anion during TiO2 photocatalytic reactions

The one-electron reduction of methyl viologen (MV(2+)) mediated by the carbon dioxide radical anion (CO(2)(*-)) during photocatalytic reactions in a colloidal TiO(2) aqueous solution (pH 2) has been investigated by time-resolved absorption spectroscopy. The formation of MV(*+) generated from the one-electron reduction reaction with CO(2)(*-), which is generated from the one-electron oxidation reactions with the photogenerated holes (h(+)), was directly observed. The spectral features of the photogenerated charge carriers and the kinetic analysis of the formation process of MV(*+) revealed that the CO(2)(*-), desorbed from the surface, reacts with MV(2+) via a homogeneous electron-transfer process in the bulk solution.     

Pulse radiolysis studies on galactose oxidase

Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu(II), no Tyr(*) radical) reacts with pulse radiolysis generated formate radicals CO(2)(*-) to give an intermediate UV-vis spectrum assigned as RSSR(*-), peak at 450 nm (epsilon = 8100 M(-1) cm(-1)). From a detailed kinetic analysis at 450 nm, pH 7.0, the following steps have been identified. First the strongly reducing CO(2)(*-) (-1.9V) reduces GOase(semi) (k(0) > or = 6.5 x 10(8) M(-1) s(-1)) to a species GOase(semi)(*-). This is followed by biphasic reactions (i) GOase(semi)(*-) + GOase(semi) (k(1) = 1.6 x 10(7) M(-1) s(-1)) to give GOase(semi) + P(*-) and (ii) P(*-) + GOase(semi) (k(2) = 6.7 x 10(6) M(-1) s(-1)) to give GOase(semi)RSSR(*-). There are no significant absorbance changes for the formation of GOase(semi)(*-) and P(*-), which are Cu(I) (or related) species. However, GOase(semi)RSSR(*-) has an absorption spectrum which differs significantly from that of GOase(semi). The 450 nm peak is characteristic of an RSSR(*-) radical with two cysteines in close sequence proximity and is here assigned to Cys515-Cys518, which is at the GOase surface and 10.2 A from the Cu. On chemical modification of the RSSR group with HSPO(3)(2-) to give RSSPO(3)H(-) and RS(-), absorbance changes are approximately 50% of those previously observed. The decay of RSSR(*-) (0.17 s(-1)) results in the formation of GOase(red). No RSSR(*-) formation is observed in the reaction of GOase(semi) Tyr495Phe with CO(2)(*-), and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(ox) with CO(2)(*-), a single-stage reaction gives GOase(semi).     

Photosensitizing potential of ciprofloxacin at ambient level of UV radiation

Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV-R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L-929 and NIH-3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2*-) and hydroxyl radical (*OH) was studied under the exposure of ambient intensities of UV-A (1.14, 1.6 and 2.2 mW cm(-2)), UV-B (0.6, 0.9 and 1.2 mW cm(-2)) and sunlight (60 min). The drug was generating 1O2, O2*- and *OH in a concentration and dose-dependent manner. Sodium azide (NaN3) and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90-95% O2*- generation. The drug (5-40 microg mL(-1)) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL(-1)) confirms the involvement of ROS in drug-induced lipid peroxidation. The generation of *OH radical was further confirmed by using specific quenchers of *OH such as mannitol (0.5 M) and sodium benzoate (0.5 M). 2'-deoxyguanosine (2'-dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin-sensitized photo-degradation of guanine base. L-929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 microg mL(-1)) under UV-A, UV-B and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3 cell line at higher drug concentration (300 microg mL(-1)) showed a decrease in cell viability by 54%, 56% and 59% under UV-A, UV-B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L-929 cell line were in accordance with MTT assay. The NIH-3T3 cell line showed a higher photosensitizing potential than L-929. The phototoxicity end point shows a time- and concentration-dependent statistically significant (P<0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV-induced photo-peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin-induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.     

Photoinduced long-lived charge separation in a tetrathiafulvalene-porphyrin-fullerene triad detected by time-resolved electron paramagnetic resonance

Photoinduced electron transfer has been observed in a molecular triad, consisting of a porphyrin (P) covalently linked to a tetrathiafulvalene (TTF) and a fullerene derivative (C(60)), in the different phases of the liquid crystal E-7 and in a glass of 2-methyltetrahydrofuran (2-MeTHF) by means of time-resolved electron paramagnetic resonance (EPR) spectroscopy. In both solvents, an EPR signal observed immediately after excitation has been assigned to the radical pair TTF(*+)-P-C(60)(*-), based on its magnetic interaction parameters and spin polarization pattern. In the 2-MeTHF glass and the crystalline phase of E-7, the TTF(*+)-P-C(60)(*-) state is formed from the TTF-(1)P-C(60) singlet state via an initial TTF-P(*+)-C(60)(*-) charge-separated state. Long-lived charge separation ( approximately 8 mus) for the singlet-born radical pair is observed in the 2-MeTHF glass at cryogenic temperatures. In the nematic phase of E-7, a high degree of ordering in the liquid crystal is achieved by the molecular triad. In this phase, both singlet- and triplet-initiated electron transfer routes are concurrently active. At room temperature in the presence of the external magnetic field, the triplet-born radical pair (T)(TTF(*+)-P-C(60)(*-)) has a lifetime of approximately 7 mus, while that of the singlet-born radical pair (S)(TTF(*+)-P-C(60)(*-)) is much shorter (<1 mus). The difference in lifetimes is ascribed to spin dynamic effects in the magnetic field.     

Effect of structural modification on photodynamic activity of hypocrellins

Aluminum ion complexed 5,8-di-Br-hypocrellin B is a new water-soluble perylenequinonoid derivative with enhanced absorption over hypocrellin B (HB) in the phototherapeutic window (600-900 nm). Electron paramagnetic resonance and 9,10-diphenyl-anthracene bleaching methods were used to investigate the photosensitizing activity of [AL2(5,8-di-Br-HB)Cl4]n in the presence of oxygen. Singlet oxygen, superoxide anion radical and hydroxyl radical can be generated by [AL2(5,8-di-Br-HB)CL4]n photosensitization. Singlet oxygen (1O2) is formed via energy transfer from triplet-state [AL2(5,8-di-Br-HB)CL4]n to ground-state molecular oxygen. 1O2 participates in the generation of a portion of superoxide anion radical (O2.-). Besides superoxide anion radical (O2.-) may originate from the electron transfer between the triplet-state [AL2(5,8-di-Br-HB)CL4]n and the ground-state molecular oxygen. OH is formed through the Fenton-Haber-Weiss reaction and the decomposition of DMPO-1O2 adduct. Compared with HB [AL2(5,8-di-Br-HB)CL4]n primarily remains and enhances the generation efficiency of superoxide anion radical and hydroxyl radical but that of singlet oxygen decreases.