ON THE ACRIDINE AND THIAZINE DYE SENSITIZED PHOTODYNAMIC INACTIVATION OF LYSOZYME—SINGLET OXYGEN SELF-QUENCHING BY THE SENSITIZERS

Abstract— The quantum yield of the photodynamic inactivation of lysozyme increases in the sequence acridine orange, methylene blue, proflavine and acriflavine (1:5:6:12). At least up to protein concentrations of 0.1 m M , singlet oxygen is exclusively responsible for the inactivation of the enzyme. For methylene blue, acriflavine and proflavine the quantum yields decrease considerably with increasing dye concentrations. From measurements in H₂O and D₂O buffer solutions it was concluded that in the case of methylene blue the effect is mainly caused by the quenching of singlet oxygen [rate constant (3–4) × 10⁸ M ⁻¹ s⁻¹]. For the acridine sensitizers both singlet oxygen and dye triplet quenching processes have to be taken into consideration. It has been found that all sensitizers act as competitive inhibitors of the enzymatic reaction of lysozyme. However, the dye-protein interaction near the active center cannot be responsible for the observed dye self-quenching effect.     

OXYGEN EFFECT IN THE PROTECTION OF E. COLI AGAINST U.V. INACTIVATION AND MUTAGENESIS BY ACRIDINE ORANGE*

Abstract— Protection by acridine orange against ultraviolet light effects in resting cells of E. coli B/r/1, try^- was studied with special reference to a possible oxygen effect. Dose-response relationships were described by the function S= 1–(1 - e-^kD)ⁿ where S is the surviving fraction and D is the u.v. dose in ergs/mm². For cells suspended in 5 × 10^--⁶M acridine orange (AO) in air, the radiation sensitivity k was reduced from 0.010 (ergs/mm²)^)-1 in the absence of the dye to 0.0053 (ergs/mm²)^-1 in the presence of the dye. Under anoxia at this AO concentration, k was further reduced to 0.0015 (ergs/mm²)^-1. The oxygen effect ratio, kO₂/k_N2, was 3.5 at this concentration of AO. Greater protection was observed in cells suspended in 2 × 10^--⁵M AO, the oxygen effect ratio was unchanged. No oxygen effect was detected in the u.v. response in the absence of the dye. The value of n was reduced from about 12 with no dye to about 5 at dye concentrations of 5 × 10^--⁶M AO or more when oxygen was present. Under anoxia, in the presence of AO, n was further reduced to about 1.3. Atebrin, an efficient u.v. protective agent but an inefficient photodynamic agent, had no oxygen effect for protection against u.v. inactivation. Acridine orange protected against u.v.-induced reversion to tryptophan indepence in E. coli WP2 to about the same extent as it did for inactivation. A similar oxygen effect was observed for both inactivation and mutagenesis.     

EVIDENCE FOR THE PRODUCTION OF SINGLET OXYGEN BY PHOTOEXCITED ACRIDINE ORANGE

Abstract In contrast to previous results obtained with the nitroxide radical detection technique, generation of the specific ¹O₂ oxidation product of cholesterol shows that photoexcited acridine orange produces singlet oxygen.     

COMPARATIVE STUDY OF OXIDATION OF NUCLEIC ACID COMPONENTS BY HYDROXYL RADICALS, SINGLET OXYGEN AND SUPEROXIDE ANION RADICALS

Abstract— Superoxide radicals, singlet oxygen and hydroxyl radicals are individually or in combination involved in radiation or photochemical processes and in various enzymatic reactions. The reactivity and the mechanism of reaction of these oxygen species with some biologically significant DNA components were investigated through the characterization of the final oxidation products.
Superoxide radicals appear to be unreactive with purine and pyrimidine 2'-deoxyribonucleosides. However, the autoxidation reaction of 6-hydroxydopamine leads to extensive degradation of thymine through the intermediary of hydroxyl radicals. Chemically and microwave-discharge generated singlet oxygen oxidation is specific to 2'-deoxyguanosine. The main oxidized products of these reactions were also characterized as well as an as yet unidentified nucleoside in the methylene-blue photooxydation of 2-deoxyguanosine. These results, in addition to specific deuterium effect experiments, lend support to the involvment of singlet oxygen (type II mechanism) in the methylene-blue photosentization. No singlet oxygen effect was observed in aqueous irradiated system.     

溴酸钾氧化吖啶黄催化褪色光度法测定痕量亚硝酸根的研究

研究了在 0.10 mol·L-1的盐酸介质中,亚硝酸根催化溴酸钾氧化伯胺基结构碱性染料吖啶黄褪色反应的适宜条件与影响因素,建立了催化动力学褪色光度法测定痕量亚硝酸盐的新方法。亚硝酸根浓度在0.25~20.00μg/25 mL范围内符合比耳定律,其λmax=455nm,Sandell灵敏度为36.20 ng·cm-2,RSD为1.15%~5.97%,回收率为 97.5%~105.0%。方法用于环境样品、生物材料、食品等实际样品中亚硝酸根的测定,试剂简单,方法快速,选择性好,结果满意。     

SUPEROXIDE RADICALS, SUPEROXIDE DISMUTASES and OXYGEN TOXICITY IN PLANTS

Abstract In plants, as in other aerobic organisms, O₂⁻ is a commonly encountered intermediate of oxygen reduction and superoxide dismutases provide a defense against the potential cytotoxicity of this radical. The superoxide dismutases found in plants resemble those encountered in other organisms. Within chloroplasts one finds the CuZn enzyme, while mitochondria contain the Mn enzyme. Nymphaceae, ginkoaceae and cruciferae are unusual among plants, indeed among eukaryotes, in that they contain an iron superoxide dismutase. Bipyridylium herbicides, such as paraquat, exert their effect by diverting electron flow from photosystem I and thus increasing 0₂– production. Paraquat-resistant genotypes of horseweed, tobacco and rye grass were found to contain elevated superoxide dismutase. This enzyme also appears to provide a defense against sulfur dioxide, sunscald and photooxidative death. The evidence supporting these statements and possible explanations are discussed.     

INACTIVATION OF CELL SURFACE RECEPTORS BY PHEOPHORBIDE a, A GREEN PIGMENT ISOLATED FROM Psychotria acuminata

Abstract— The inhibition of cytokine and monoclonal antibody binding to cell surfaces caused by an extract of Psychotria acuminata, a medicinal plant used in the traditional medicine of the people of Belize (Central America), was attributed to the presence of pheophorbide a and pyropheophorbide a Since the binding of tumor necrosis factor-alpha, interleukin-8, complement factor 5a as well as epidermal growth factor to target cells was dramatically reduced, the inhibition was not receptor or cytokine specific. In addition, the respective binding of radiolabeled monoclonal antibodies CL203 and R15.7 to the cell surface antigens intracellular cell adhesion molecule-1 and lymphocyte function-associated antigen-1 ß-chain was decreased by pretreatment of cells with pheophorbide a as well. In all cases, the inhibition by pheophorbides was dependent on the simultaneous presence of light, indicating causative involvement of a photodynamic process. These observations are not unique to pheophorbides and can be extended to porphyrins as well as to other photodynamic agents. Cytotoxicity resulting from photodynamic therapy (PDT) has been documented by many studies. Our investigations suggest that the inactivation of cell surface receptors contributes not only to the antitumor effect of PDT but also to the systemic immunosuppression, a serious side effect of PDT.     

TWO-DIMENSIONAL DISTRIBUTIONS OF TEMPERATURE AND OH, C_2 AND CH RADICALS IN FLAME DETECTED BY LASERINDUCED FLUORESCENCE

The in situ, high spatial resolution, two-dimensional distributions of OH(X), C_2(X) and CH(X) radicals in premixed paraffin/air flames are first reported. The measurements were done with the laser-induced fluorescence spectroscopies (LIFS) of the transitions of OH(A~Σ~+←→X~2Π), C_2(A~3Π←→X~3Π) and CH(B~2Σ←→X~2Π), (A~2△→X~2Π). The results show that OH(X) populated the edge of the outer flame, while C_2(X) and CH(X) the edge of the inner cone of the flame.The local rotational temperature of OH radical for a small zone was detected by the saturated laser-induced fluorescence spectroscopy of A~2Σ~+, v′=0←→X~2Π, v=″0 transition. The two-dimensional temperature distribution for the flame was measured through moving the position of the burner.     

INHIBITORY EFFECTS OF ETHANOL ON THE PHOTODYNAMIC CELL INACTIVATION AND PETITE INDUCTION BY EUFLAVINE IN YEAST, Saccharomyces cerevisiae

Abstract— Effects of active oxygen scavengers on cell inactivation and petite induction of yeast by the photodynamic action of euflavine were examined. Histidine, sodium azide, 1,3-diphenyl-isobenzofuran and p-carotene, which are singlet oxygen scavengers, inhibited photodynamic cell killing. Histidine and sodium azide inhibited petite induction, too. These results suggested that photobiological effects of euflavine are induced via singlet oxygen-mediated Type II reaction process. In this work, however, we found that ethanol, which is reported to be a hydroxyl radical scavenger, notably inhibited photodynamic cell inactivation and petite induction by euflavine. Inhibition of petite induction was increased with increasing concentration of ethanol. Decrease of absorbance of euflavine by irradiation was also inhibited by the addition of ethanol.
These results suggested that ethanol possibly acts as a singlet oxygen scavenger.     

CUTANEOUS PHOTOTOXICITY OF TETRACYCLINE ANTIBIOTICS: GENERATION OF FREE RADICALS and SINGLET OXYGEN DURING PHOTOLYSIS AS MEASURED BY SPIN-TRAPPING and THE PHOSPHORESCENCE OF SINGLET MOLECULAR OXYGEN

Chlortetracycline (CTC) generated an aryl radical in aqueous buffer (pH 7.4) during near UV irradiation, as evidenced by the formation of 2-methyl-2-nitrosopropane spin adducts. The radical was produced via dechlorination, a photoprocess not previously reported for tetracyclines. Demeclocyc-line (DEM), another chlorinated tetracycline, did not produce detectable aryl radicals. Relative ¹O₂ yields obtained by direct luminescence measurements at 1268 nm for five tetracyclines in alkaline ethanol (demeclocycline · tetracycline · chlortetracycline · doxycycline · minocycline) showed that DEM produced approximately three times as much singlet oxygen as CTC. This constitutes direct evidence that tetracyclines sensitize both Type I and Type II photoreactions.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

HYDROGEN and OXYGEN PHOTOPRODUCTION BY MARINE ALGAE*

Abstract The first measurements of the simultaneous photoproduction of hydrogen and oxygen in marine green algae are reported. Eight species in the genera Chlamydomonas, Chlorella and Halochlorocococcum were tested in CO₂-free seawater. Four of the five species of Chlamydomonas were able to produce hydrogen in the light after a period of 3 - 4 h of dark anaerobic adaptation. Only one of the two Chlorella species tested was able to photoproduce hydrogen, in trace amounts. Halochlorocococcum fla–9 gave positive results and Chlamydomonas species (clone f-9) had a steady-state rate of hydrogen and oxygen production during irradiation with a stoichiometric ratio near 2:1. The integrated yields of hydrogen and oxygen produced by this species corresponds to about 450 turnovers of the photochemical reaction centers. This number exceeds (by about a factor of 20) the electron-carrying capacity of the electron transport chain linking Photosystems I and II. These data suggest that Chlamydomonas f-9 makes seawater a potential substrate for solar hydrogen and oxygen production.     

Bond dissociation energies and radical heats of formation in CH3Cl,CH2Cl2, CH3Br,CH2Br2, CH2FCl,and CHFCl2

An analysis of thermochemical and kinetic data on the bromination of the halomethanes CH_4–nX_n (X = F, Cl, Br; n = 1–3), the two chlorofluoromethanes, CH₂FCl and CHFCl₂, and CH₄, shows that the recently reported heats of formation of the radicals CH₂Cl, CHCl₂, CHBr₂, and CFCl₂, and the C? H bond dissociation energies in the matching halomethanes are not compatible with the activation energies for the corresponding reverse reactions. From the observed trends in CH₄ and the other halomethanes, the following revised ΔH°_f,298 (R) values have been derived: ΔH°_f(CH₂Cl) = 29.1 ± 1.0, ΔH°_f(CHCl₂) = 23.5 ± 1.2, ΔH_f(CH₂Br) = 40.4 ± 1.0, ΔH°_f(CHBr₂) = 45.0 ± 2.2, and ΔH°_f(CFCl₂) = ?21.3 ± 2.4 kcal mol^?1. The previously unavailable radical heat of formation, ΔH°_f(CHFCl) = ?14.5 ± 2.4 kcal mol^?1 has also been deduced. These values are used with the heats of formation of the parent compounds from the literature to evaluate C? H and C? X bond dissociation energies in CH₃Cl, CH₂Cl₂, CH₃Br, CH₂Br₂, CH₂FCl, and CHFCl₂.     

The elusive methene ylides CH2CIH,CH2FH and CH2OH2

The possible existence of the ylides CH₂CIH, CH₂₅FH and CH₂OH₂ as stable neutral species in the gas phase has been investigated by the neutralization–reionization (NR) mass spectrometry of their radical cations using a double-focusing mass spectrometer of reversed geometry. The experiments were, for the most part, performed under single-collision conditions with Xe as the neutralization target gas and He and O₂ as reionization agents. For each ylidion a peak was observed in their NR mass spectrum which indicated that the neutral ylide had apparently been produced. However for CH₂FH⁺˙ and CH₂OH₂⁺˙ the m/z 34 and m/z 32 peaks, respectively, were attribut-able to interferences from the natural isotopic abundance of ions of lower mass. For CH₂CIH⁺˙, the NR recovery signal was found to arise from the presence of CH₃CI⁺˙ as an impurity in the ylidion flux. This was proved by examination of the collisional activation mass spectra of the [C, H₃, CI]⁺˙ ions produced in the NR mass spectra of the conventional ions and ylidions, an experiment performed using a triple-sector mass spectrometer.     

Study of the interaction between aniline and CH3CN, CH3Cl and CH3F

A computational study of dimers formed by aniline and one or two CH₃X molecules, X being CN, Cl or F, was carried out to elucidate the main characteristics of the interacting systems. Two different structures were found for each of the dimers, depending on the relative location of the CH₃X molecule with respect to the NH₂ hydrogen atoms. The most stable complex is formed with acetonitrile, with a complexation energy amounting to ?27.0?kJ/mol. Methyl chloride and methyl fluoride form complexes with complexation energies amounting to ?18.1 and ?17.5?kJ/mol, respectively, though the structural arrangement is quite different for both structures. In most complexes, the leading contribution to the stabilization of the complex is dispersion, though the electrostatic contribution is almost as important. Three different minima were obtained for clusters containing two CH₃X molecules depending on the side they occupy with respect to the phenyl ring. The complexation energies for these structures amount to ?58.5, ?38.6 and ?36.3?kJ/mol for acetonitrile, methyl chloride and methyl fluoride, respectively.     

FTIR spectra of HSCH2CH2SH,CH3SCH2SH,and CH3SSCH3 in argon and nitrogen matrices

Matrix-isolated IR spectra of 1,2-ethanedithiol, (methylthio)methanethiol, and dimethyldisulfide were recorded from 400 to 4000 cm⁻¹ in argon and nitrogen matrices at 12 K. The appearances of new bands around the vibrations of the monomers provide evidence for the presence of dimer and multimers of 1,2-ethanedithiol and (methythio)methanethiol in argon and nitrogen matrices. The co-existence of two different dimers of (methylthio)methanethiol in nitrogen matrices is considered on the basis of the experimental data. The spectroscopic data confirm the presence of one gauche and one trans conformer for 1,2-ethanedithiol and at least three conformers for (methylthio)methanethiol, but only one gauche form for dimethyl disulfide in the matrices.     

A computational exploration of some transnitrosation and thiolation reactions involving CH3SNO, CH3ONO and CH3NHNO

Nitric oxide (NO) is a biologically active species and its carrier molecules RXNO (X = S, O, NH) have drawn significant attention recently. In the present work, the CBS-QB3 level of theory was used to study the transnitrosation and thiolation reaction between MeXNO (X = S, O, and NH) molecules and three reactive forms of the methanethiol: the neutral molecule, MeSH, the anion, MeS-, and the radical, MeS . The transnitrosation and thiolation reactions between MeXNO and MeSH have the highest barriers, both with and without a molecule of water assisting. Reactions with MeS- proceed with much lower barriers, while reactions with radical MeS have the lowest barriers. Comparing the reactions of MeXNO (X = S, O, NH), both transnitrosation and thiolation are more favorable for X = S than X = O or NH.     

Energetics of the O-H bond and of intramolecular hydrogen bonding in HOC6H4C(O)Y (Y = H, CH3, CH2CH=CH2, C[triple bond]CH, CH2F, NH2, NHCH3, NO2, OH, OCH3, OCN, CN, F, Cl, SH, and SCH3) compounds

The energetics of the phenolic O-H bond in a series of 2- and 4-HOC 6H 4C(O)Y (Y = H, CH3, CH 2CH=CH2, C[triple bond]CH, CH2F, NH2, NHCH 3, NO2, OH, OCH3, OCN, CN, F, Cl, SH, and SCH3) compounds and of the intramolecular O...H hydrogen bond in 2-HOC 6H 4C(O)Y, was investigated by using a combination of experimental and theoretical methods. The standard molar enthalpies of formation of 2-hydroxybenzaldehyde (2HBA), 4-hydroxybenzaldehyde (4HBA), 2'-hydroxyacetophenone (2HAP), 2-hydroxybenzamide (2HBM), and 4-hydroxybenzamide (4HBM), at 298.15 K, were determined by micro- or macrocombustion calorimetry. The corresponding enthalpies of vaporization or sublimation were also measured by Calvet drop-calorimetry and Knudsen effusion measurements. The combination of the obtained experimental data led to Delta f H m (o)(2HBA, g) = -238.3 +/- 2.5 kJ.mol (-1), DeltafHm(o)(4HBA, g) = -220.3 +/- 2.0 kJ.mol(-1), Delta f H m (o)(2HAP, g) = -291.8 +/- 2.1 kJ.mol(-1), DeltafHm(o)(2HBM, g) = -304.8 +/- 1.5 kJ.mol (-1), and DeltafHm(o) (4HBM, g) = -278.4 +/- 2.4 kJ.mol (-1). These values, were used to assess the predictions of the B3LYP/6-31G(d,p), B3LYP/6-311+G(d,p), B3LYP/aug-cc-pVDZ, B3P86/6-31G(d,p), B3P86/6-311+G(d,p), B3P86/aug-cc-pVDZ, and CBS-QB3 methods, for the enthalpies of a series of isodesmic gas phase reactions. In general, the CBS-QB3 method was able to reproduce the experimental enthalpies of reaction within their uncertainties. The B3LYP/6-311+G(d,p) method, with a slightly poorer accuracy than the CBS-QB3 approach, achieved the best performance of the tested DFT models. It was further used to analyze the trends of the intramolecular O...H hydrogen bond in 2-HOC 6H 4C(O)Y evaluated by the ortho-para method and to compare the energetics of the phenolic O-H bond in 2- and 4-HOC 6H 4C(O)Y compounds. It was concluded that the O-H bond "strength" is systematically larger for 2-hydroxybenzoyl than for the corresponding 4-hydroxybenzoyl isomers mainly due to the presence of the intramolecular O...H hydrogen bond in the 2-isomers. The observed differences are, however, significantly dependent on the nature of the substituent Y, in particular, when an intramolecular H-bond can be present in the radical obtained upon cleavage of the O-H bond.     

Formation and hydrolysis of gas‐phase [UO2(R)]+: R═CH3, CH2CH3, CH═CH2, and C6H5

The goals of the present study were (a) to create positively charged organo‐uranyl complexes with general formula [UO₂(R)]⁺ (eg, R═CH₃ and CH₂CH₃) by decarboxylation of [UO₂(O₂C─R)]⁺ precursors and (b) to identify the pathways by which the complexes, if formed, dissociate by collisional activation or otherwise react when exposed to gas‐phase H₂O. Collision‐induced dissociation (CID) of both [UO₂(O₂C─CH₃)]⁺ and [UO₂(O₂C─CH₂CH₃)]⁺ causes H⁺ transfer and elimination of a ketene to leave [UO₂(OH)]⁺. However, CID of the alkoxides [UO₂(OCH₂CH₃)]⁺ and [UO₂(OCH₂CH₂CH₃)]⁺ produced [UO₂(CH₃)]⁺ and [UO₂(CH₂CH₃)]⁺, respectively. Isolation of [UO₂(CH₃)]⁺ and [UO₂(CH₂CH₃)]⁺ for reaction with H₂O caused formation of [UO₂(H₂O)]⁺ by elimination of ·CH₃ and ·CH₂CH₃: Hydrolysis was not observed. CID of the acrylate and benzoate versions of the complexes, [UO₂(O₂C─CH═CH₂)]⁺ and [UO₂(O₂C─C₆H₅)]⁺, caused decarboxylation to leave [UO₂(CH═CH₂)]⁺ and [UO₂(C₆H₅)]⁺, respectively. These organometallic species do react with H₂O to produce [UO₂(OH)]⁺, and loss of the respective radicals to leave [UO₂(H₂O)]⁺ was not detected. Density functional theory calculations suggest that formation of [UO₂(OH)]⁺, rather than the hydrated U^VO₂⁺, cation is energetically favored regardless of the precursor ion. However, for the [UO₂(CH₃)]⁺ and [UO₂(CH₂CH₃)]⁺ precursors, the transition state energy for proton transfer to generate [UO₂(OH)]⁺ and the associated neutral alkanes is higher than the path involving direct elimination of the organic neutral to form [UO₂(H₂O)]⁺. The situation is reversed for the [UO₂(CH═CH₂)]⁺ and [UO₂(C₆H₅)]⁺ precursors: The transition state for proton transfer is lower than the energy required for creation of [UO₂(H₂O)]⁺ by elimination of CH═CH₂ or C₆H₅ radical.