Photorelease Dynamics of Nitric Oxide from Cysteine-Bound Roussin's Red Ester.

Nitric oxide (NO) can either boost or impede the growth of cancer cells depending on its concentration. Therefore, any anticancer treatment using NO requires precisely controlled NO administration to the target cells in terms of dosage and timing. In this context, photochemically activated NO donors were actively explored, but their detailed NO-releasing dynamics, which is crucial for their use, is not known yet. We determined detailed photoexcitation dynamics of a stable, nontoxic, and water-soluble NO precursor, cysteine-bound Roussin's Red Ester (Cys-RRE), including secondary reactions of the nascent photoproducts. The primary quantum yields of the NO dissociation from the photoexcited Cys-RRE were found to be 24-54% depending on the excitation wavelength; however, the geminate rebinding of NO with the nascent radical reduced the level of biologically available NO to as low as 12%. Such information is useful to achieve efficient NO delivery to practical chemical and biological targets.

Controlled release of HNO from chemical donors for biological applications.

Nitroxyl (HNO) is a small molecule with various pharmacological effects, including cardioprotective action. It is thought to serve as a modulator of various biochemical pathways. But, it is difficult to apply HNO directly for biological experiments or therapeutic treatment because it is highly reactive, readily dimerizing or reacting with biological targets under ambient conditions. Therefore, HNO donor molecules that release HNO under physiological conditions, especially those that allow precisely controllable release, would be useful to study the activities of HNO at the cellular level. This short review focuses on recently developed photocontrollable HNO-releasing compounds, which are expected to be suitable for achieving site-specific and temporally controlled HNO release in biomedical investigations. An illustrative application for the study of HNO-mediated upregulation of calcitonin gene-related peptide (CGRP) in A549 cells is described.

Photodynamic efficacy of chlorin p6: a pH dependent study in aqueous and lipid environment.

Photodynamic efficacy of chlorin p6, a potential candidate of photodynamic therapy (PDT), has been studied at pH 5.0, 6.0 and 7.6 in aqueous and lipid environment. Increased chlorin p6 mediated photodynamic bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a measure of singlet oxygen yield, was obtained at higher pH. Rate of photodynamic bleaching of RNO was also higher at higher pH and the rate decreased with lowering in pH of irradiated solution. Photodynamic oxidation of tryptophan was also found to be higher at higher pH. Diminished oxidation of RNO was obtained with decrease in pH of irradiated solution. Both, RNO bleaching and tryptophan oxidation was significantly reduced by sodium azide, a known quencher of singlet oxygen. At lower pH, chlorin p6 mediated photodynamic malondialdehyde (MDA) and lipid hydroperoxide formation in egg lecithin liposome was higher. At higher pH chlorin p6 was found to be photodynamically more effective in aqueous environment whereas at lower pH chlorin p6 was photodynamically more effective in hydrophobic environment.

Photochemistry of the [Fe4(mu3-S)3(NO)7]- complex in the presence of S-nucleophiles: a spectroscopic study.

Biological systems usually contain cysteine, glutathione or other sulfur-containing biomolecules. These S-nucleophiles were found to affect drastically the [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photolysis pathway generating products completely different from that of the neat cluster, which produces Fe(II) and NO and S(2-). The effect is interpreted in terms of formation of a pseudo-cubane adduct, [Fe(4)(mu(3)-S)(3)(mu(3)-SR)(NO)(7)](2-), whose existence in equilibrium with the parent complex has no detectable influence on the spectral properties, whereas shifts the redox potential and induces photoconversion leading to the Fe(III) species and N(2)O. Characteristic bond lengths, bond angles and atomic Mulliken charges were calculated using semi-empirical quantum chemical methods for the RBS anion and a series of pseudo-cubane complexes with S-donor or N-donor ligands. The results justify the hypothesis of the adduct formation and show that only in case of S-ligands the higher contribution of the Fe(III)-NO(-) components in adduct than in RBS is observed, which on excitation can undergo heterolytic cleavage yielding Fe(III) and NO(-), converted rapidly into N(2)O. These results are crucial in understanding the physiological activity of RBS. Fe(III) formation can be detected only when the S-ligand enables formation of a stable Fe(III) compound; the effect was recorded in the presence of sulfide, thioglycolate, 2-mercaptopropionate, mercaptosuccinate, penicillamine, 2,3-dimercaptosuccinate, 2,3-dimercaptopropanol, and thiocyanate. For all these S-ligands the Fe(III) photoproducts were identified and characterised. In the case of other thiolates, their excess results in fast reduction of Fe(III) to Fe(II), whereas N(2)O can be still detected. Quantum yields of Fe(III) formation in the presence of the S-ligands are considerably higher than that of the Fe(II) photoproduction from neat [Fe(4)(mu(3)-S)(3)(NO)(7)](-).

Visible light induced reversible extrusion of nitric oxide from a ruthenium(II) nitrosyl complex: a facile delivery of nitric oxide.

The new ruthenium compound [Ru(NO)(pysiS4)]Br (3) (pysiS4 = 2,6-bis(3-triphenylsilyl-2-sulfanylphenylthiomethyl)pyridine), containing sterically bulky SiPh3 groups ortho to the thiolate donors, has been synthesized. In solution, 3 releases NO efficiently on exposure to visible light (lambda >/= 455 nm) at room temperature to afford [Ru(Br)(pysiS4)] (4). Treatment of 4 with NO yielded exclusively 3 without any metal-bound side reaction.

NO-dependent phototoxicity of Roussin's black salt against cancer cells.

A metal-nitrosyl complex, Roussin's black salt (RBS), releases nitric oxide after illumination. Approximately 3.7 NO molecules were released from one RBS molecule. Both short- and long-term effects of photogenerated NO on the two neoplastic cell lines: human (SK-MEL188) and mouse (S91) have been investigated. Exogenous NO from RBS was toxic to cells in a dose-dependent manner. Apoptotic damage predominates in the response to the injury, as shown by TUNEL assay. NO and its short-lived metabolites, but not other RBS photoproducts, are responsible for cellular death. RBS in dark was toxic to cells at concentrations above 1 microM. This relatively high cytotoxicity of RBS in the dark prevents its application as a systemic anticancer agent in vivo, unless it is applied locally.

3-alkyl- and 3-aryl-(7-oxo-7H-furo[3, 2-g]chromen-5-yl)alkanoic acids as inhibitors of leukotriene B4 biosynthesis.

The synthesis and biological properties of 3-alkyl-and 3-aryl-(7-oxo-7H-furo [3, 2-g]chromen-5-yl)alkanoic acids are described. The compounds were evaluated for their ability to inhibit soybean 15-lipoxygenase (15-LO) and the production of leukotriene B(4) (LTB(4)) in bovine polymorphonuclear leukocytes. The furocoumarins were further investigated for their lipophilicity and their capacity to photobleach N, N-dimethyl-p-nitrosoaniline (RNO) after irradiation with UVA light. The synthesised furocoumarins showed only a moderate ability to inhibit the 15-LO and the production of LT B(4). The 3-alkyl substituted furocoumarins had a higher capacity to photobleach RNO than then the 3-aryl substituted furocoumarinso. The inhibitory activity of the newcompounds was quite modest in comparison to other known inhibitors of 15-LO and LTB(4) production. Nevertheless, they may provide the basis for the development of more potent antiinflammatory potential photoreactive furocoumarins.

Analysis of nanomolar S-nitrosothiol concentrations in physiological media.

S-Nitrosothiols occur endogenously and are thought to function as storage forms and/or stable carriers of nitric oxide. Moreover, the S-nitrosothiols have been postulated to function as neurotransmitters and mediate the vasodilator action of glyceryl trinitrate. Because of the increasing pharmacological and physiological interest in S-nitrosothiols, a sensitive method for analysis of these substances is required. We describe a sensitive method based on adsorptive stripping voltammetry for measurement of two S-nitrosothiols, namely, S-nitroso-N-acetyl-D-penicillamine (SNAP) and S-nitrosoglutathione (SNOG), in Krebs' solution. The method is based on the irreversible electrochemical reduction of SNAP and SNOG at the hanging mercury drop electrode (HMDE). The analytes were adsorbed at the HMDE for 60 s at -0.100 V, then exposed to a cathodic linear potential scan of 100 mV s(-10) which resulted in the reduction of SNAP at -0.470 V and SNOG at -0.530 V. Under these conditions, 4 nM SNAP and 11 nM SNOG were readily quantified. Using the above method, we were able to confirm the rapid degradation of SNAP under UV irradiation. Reproducibility of the method as applied to the analysis of these S-nitrosothiols in Krebs' solution was demonstrated by the within-day and day-to-day coefficients of variation of 1.5% and 2.0%, respectively.

Control of NO concentration in solutions of nitrosothiol compounds by light.

We studied the thermal and photolytic decomposition of two S-nitrosothiols, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), in water or propanol solutions. A "concentration clamp" (relatively constant concentration of NO as a function of time) could be implemented in a closed volume by varying the pH, concentration of nitrovasodilator and intensity of the light source. Depending on the conditions, the light either stimulated NO release or sharply decreased NO concentration in the test solutions. Changes in the absorption spectra of GSNO solutions were monitored as a function of light exposure. Generation of superoxide as a product of a photolytic decomposition reaction of S-nitrosothiols and further oxidation of NO is the most likely mechanism for light suppression of NO concentration.

Synergistic action of N-nitrosodialkylamines and near-UV in the induction of chromosome aberrations in Chinese hamster lung fibroblasts in vitro.

N-Nitrosodialkylamines are promutagens and proclastogens, requiring metabolic activation for their actions. Previously, we showed that direct-acting bacterial mutagens can be formed from N-nitrosodialkylamines on exposure to near-UV. We have now found that N-nitrosodialkylamines with near-UV irradiation are clastogenic to Chinese hamster lung cells. When the cells in culture were irradiated with near-UV for 3 h in the presence of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP) or N-nitrosomorpholine (NMOR), and then further incubated for a total period of 24 h with the N-nitrosodialkylamines, chromosome aberrations were induced. Neither the N-nitrosodialkylamine nor near-UV alone were clastogenic. Severe clastogenicity (> 50% of cells examined showing aberrations) was observed for 0.5 mM NDEA, NPYR and NPIP. The order of the clastogenic activity was NDEA, NPYR > NPIP, NDMA > NMOR. This order differed from that of bacterial mutagenicity previously reported for these N-nitrosodialkylamines plus near-UV, in which NMOR gave the strongest activity. The chromosome aberrations induced by the NPYR and NDEA plus near-UV in CHL-cells were inhibited by superoxide dismutase, glutathione and L-cysteine. Dimethylsulfoxide or D-mannitol, scavengers of hydroxy radical, and L-histidine, a scavenger of single oxygen, were ineffective. These results suggest that superoxide formed by a synergistic action of an N-nitrosodialkylamine and near-UV is the cause of the chromosome aberrations observed, an assumption consistent with the known ability of superoxide to cleave DNA.

Visible light photochemical release of nitric oxide from S-nitrosoglutathione: potential photochemotherapeutic applications.

Some aspects of the physiological role of NO may be mediated by stable NO-carriers such as S-nitrosoglutathione and related S-nitrosothiols. In this report we show that irradiation of S-nitrosoglutathione at either absorption band (lambda max = 340 nm or 545 nm) results in the release of nitric oxide. Photolysis of S-nitrosoglutathione at 545 nm exhibited a quantum yield of 0.056 +/- 0.002 and was best approximated by a first-order process with kobs = 4.9 x 10(-7) +/- 0.3 x 10(-7) s-1. The photolytic release of NO from S-nitrosoglutathione resulted in an enhanced cytotoxic effect of S-nitrosoglutathione on HL-60 leukemia cells. That the cytotoxic effect of S-nitrosoglutathione was diminished by the addition of oxyhemoglobin strongly suggests that NO is the cytotoxic species. The finding that NO can be readily liberated from S-nitrosoglutathione by visible radiation indicates that the photochemical properties of this compound in the visible spectrum must be considered in order to obtain meaningful data as to its physiological role and the S-nitrosoglutathione and related compounds may find use as photochemotherapeutic agents.

A wavelength dependent mechanism for rose bengal-sensitized photoinhibition of red cell acetylcholinesterase.

A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.

Study of the mode of action of some nitrodiphenyl ethers.

Nitrosoderivatives of the nitrodiphenyl ether herbicides (nitrofen, bifenox) have been studied. UV irradiation in different organic solvents gives degradation products. In buffered aqueous media, in the presence of chloroplasts and spin traps such as DMPO, hydroxy and peroxy radicals have been characterized. In organic media and in the presence of spin traps such as DMPO, PBN, 4-POBN, solvent radicals (.CHCl2, .CCl3, .CH2 [symbol: see text]) have been formed. Nitro-derivatives have been studied under UV irradiation and in the presence of tetramethylethylene (TME), alkenylhydroxylamines are formed which autoxidize in nitroxide radicals. The formation of the stable nitroxide radical occurs in the dark process after continuous irradiation. The intensity of the signal decreases strongly when a new irradiation is applied. Radical species, with analogous ESR spectral characteristics are formed on reaction with nitrodiphenyl ethers and fatty acids. The reactivity of these herbicides in micellar media (SDS, Brij 35, and CTAB) has been investigated. The kinetics of formation of the ESR signal corresponding to the photoreduction of the nitrodiphenyl ether in the presence of TME behave differently in a micellar environment as compared to solution. The intensity of the formation of the nitroxide increases under irradiation and decreases in the dark; the rotational correlation time tau c has been determined for each type of micelle. Synthetic nitrosodiphenyl ether made by the reduction of nitrodiphenyl ether using hydrogen gas and PtO2 as a catalyst gives the corresponding amine, which is oxidized with meta-chloroperbenzoic acid (m.CPBA). The nitrosodiphenyl ether in the presence of soja azolectin liposome containing a fluorescent probe has been analysed.(ABSTRACT TRUNCATED AT 250 WORDS)

Photolysis of nitrosamines and nitrosamides at neutral pH: a spin-trap study.

A model system has been used to study the types of radicals formed on denitrosation of N-nitroso compounds. Free radicals were formed at room temperature (22 degrees-23 degrees C) and neutral pH by photolytic cleavage of N-nitroso bonds and were partially characterized following their addition to the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenyl-nitrone (PBN). Carbon-centered radical adducts were obtained during nitrosamine photolysis and nitrogen-centered radical adducts during nitrosamide photolysis. Since both the nitrosamines and nitrosamides initially form nitrogen-centered radicals on photolysis, a secondary reaction or rearrangement must occur after initial N-nitroso bond cleavage in the nitrosamines. Mechanisms are proposed to account for these results.