A Comparative Kinetic and Mechanistic Study Between Tetrahydrozoline and Naphazoline Toward Photogenerated Reactive Oxygen Species

Kinetic and mechanistic aspects of the vitamin B2 (riboflavin [Rf])-sensitized photo-oxidation of the imidazoline derivates (IDs) naphazoline (NPZ) and tetrahydrozoline (THZ) were investigated in aqueous solution. The process appears as important on biomedical grounds, considering that the vitamin is endogenously present in humans, and IDs are active components of ocular medicaments of topical application. Under aerobic visible light irradiation, a complex picture of competitive interactions between sensitizer, substrates and dissolved oxygen takes place: the singlet and triplet (³Rf*) excited states of Rf are quenched by the IDs: with IDs concentrations ca.  5.0 m m and 0.02 m m Rf, ³Rf* is quenched by IDs, in a competitive fashion with dissolved ground state oxygen. Additionally, the reactive oxygen species: O₂(¹Δ_g), O₂^•−, HO^• and H₂O₂, generated from ³Rf* and Rf ^•−, were detected with the employment of time-resolved methods or specific scavengers. Oxygen uptake experiments indicate that, for NPZ, only H₂O₂ was involved in the photo-oxidation. In the case of THZ, O₂^•−, HO^• and H₂O₂ were detected, whereas only HO^• was unambiguously identified as THZ oxidative agents. Upon direct UV light irradiation NPZ and THZ generate O₂(¹Δ_g.), with quantum yields of 0.2 (literature value, employed as a reference) and 0.08, respectively, in acetonitrile.     

ON THE GENERATION OF THE HYDROXYLATION AGENT FROM SUPEROXIDE RADICAL. CAN THE HABER–WEISS REACTION BE THE SOURCE OF OH RADICALS?

Abstract— In many biological systems, the role of O₂^- in hydroxylation and toxic processes was assumed to be due to the formation of OH radicals. The Haber-Weiss reaction (Haber and Weiss, 1934)—(H₂O₂+ O₂^-→ OH + OH^-+ O₂) was suggested as the origin of this activity.
In this study it is shown that this reaction pathway is too slow, and that OH is probably formed from the reaction of complexed superoxide with H₂O₂ or/and from the reduction of Fe(III), bound to biological compounds, by O₂^-; the reduced Fe(II) can then react with H₂O₂ as a Fenton reagent, to yield OH.
It is also shown that singlet oxygen cannot be formed in these biological systems neither from the dismutation of OJ nor from the reaction of O₂^- with OH. Singlet oxygen may be formed from the reduction of metal complexes by O₂^-.     

Comparison of the Disinfection Effects of Vacuum-UV (VUV) and UV Light on Bacillus subtilis Spores in Aqueous Suspensions at 172, 222 and 254 nm

The efficacy of UV and vacuum-UV (VUV) disinfection of Bacillus subtilis spores in aqueous suspensions at wavelengths of 172, 222 and 254 nm was evaluated. A Xe₂* excilamp, a KrCl* excilamp and a low-pressure mercury lamp were used as almost monochromatic light sources at these three wavelengths. The first-order inactivation rate constants at 172, 222 and 254 nm were 0.0023, 0.122 and 0.069 cm² mJ⁻¹, respectively. Therefore, a 2 log reduction of B .  subtilis spores was reached with fluences (UV doses) of 870, 21.6 and 40.4 mJ cm⁻² at these individual wavelengths. Consequently, for the inactivation of B .  subtilis spores, VUV exposure at 172 nm is much less efficient than exposure at the other two wavelengths, while exposure at 222 nm is more efficient than that at 254 nm, which is probably because triplet energy transfer from DPA to thymine bases at 222 nm is higher than that at 254 nm. This research indicated quantitatively that VUV light is not practicable for microorganism disinfection in water and wastewater treatment. However, in comparison with other advanced oxidation processes ( e.g. UV/TiO₂, UV/H₂O₂ or O₃/H₂O₂) the VUV-initiated photolysis of water is likely more efficient in generating hydroxyl radicals and more effective for the inactivation of microorganisms.     

INTERACTIONS OF ASCORBATE AND CHELATED IRON IN A METHYLVIOLOGEN-MEDIATED MEHLER REACTION

Abstract— –In the light, isolated spinach thylakoids consumed O₂ in the presence of methylviologen, and ascorbate was found to interact with this reaction in various ways. Chelating-resin was used to remove metal impurities from the assay medium. Ascorbate diminished the H₂0₂ pool in resin-untreated solutions, while in resin-treated solutions ascorbate had no effect on H₂O₂ concentrations. A Fenton catalyst (Fe-EDTA) increased O₂ uptake in the presence of ascorbate and decreased the amount of O₂ recovered by catalase. Ascorbate tripled the rate of the methylviologen-mediated Mehler reaction, and the O₂ consumed was liberated to 50% of its original concentration by catalase. Superoxide dismutase reversed the effects of ascorbate on the Mehler reaction rates. These results indicate that ascorbate can stimulate Mehler reactions indirectly by promoting a Fenton-type reaction as well as stimulating Mehler reactions directly by reducing 2O₂^- to 2H₂O₂. The promotion of a Fenton-type reaction by ascorbate appears to be the cause of H₂O₂ depletion in resin-untreated solutions.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

SEARCH FOR SINGLET OXYGEN LUMINESCENCE IN THE DISPROPORTIONATION OF HO2/O2

Abstract— During the reaction HO₂+ HO₂ (or O₂^-) = H₂O₂+ O₂ in aqueous solution, no luminescence in the region 620–720 nm, expected if the product O₂ were formed in a singlet state, could be detected. If any singlet O₂ is formed, its yield must be less than 10%. Faint luminescence, sometimes found at shorter wavelengths, was shown to arise from reaction of HO₂ with impurities in the reagents present.     

BIOLUMINESCENCE FROM THE REACTION OF FMN, H2O2 AND LONG CHAIN ALDEHYDE WITH BACTERIAL LUCIFERASE

Abstract— The bioluminescent oxidation of reduced flavin mononucleotide by bacterial luciferase involves a long-lived flavoenzyme intermediate whose chromophore has been postulated to be the 4a-sub-stituted peroxy anion of reduced flavin. Reaction of long chain aldehyde with this intermediate results in light emission and formation of the corresponding acid. These experiments show that the typical aldehyde-dependent, luciferase-catalyzed bioluminescence can also be obtained starting with FMN and H₂O₂ instead of FMNH₂ and O₂. We postulate that the 4a-peroxy anion intermediate is formed directly by attack of H₂O₂ on FMN. The latter may be bound to luciferase. An enzyme bound intermediate is formed which by kinetic analysis, flavin specificity for luminescence, aldehyde dependence, and bioluminescent emission spectrum appears to be identical with the species generated by reaction of FMNH, and O₂ with luciferase. The quantum yield of the H₂O₂-_- and FMN-initiated biolumlnescence is low but can be enhanced by certain metal ions, which also stimulate a chemiluminescent reaction of oxidized flavin with H₂O₂. The peak of this chemiluminescence. however, appears to be at a shorter wavelength than that (490 nm) of the bioluminescence.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

HYDROGEN AND OXYGEN PHOTOPRODUCTION BY TITANATE POWDERS

Abstract— Uncoated powders of TiO₂ or SrTiO₃ did not produce H₂ or O₂ on UV irradiation of aqueous suspensions of the powders. TiO₂ powders coated with platinum or rhodium photoproduced H₂ on irradiation (effective wavelengths 334 and 366 nm) and the reaction was stimulated by catalytic quantities of methyl viologen. The turnover numbers for H₂ production relative to TiO₂ were very low suggesting that the powders were not acting catalytically. Hydrogen production was never stoichiometric with respect to TiO₂ and the kinetics of H₂ production were first order, not zero order as would be expected for catalytic photolysis of water. Oxygen was never detected and it appears that H₂ did not arise from water photolysis but rather from oxidation of reduced sites in TiO₂. A rhodium-coated SrTiO₃ powder prepared photochemically produced both H₂ and O₂ on irradiation but the turnover numbers were very low. A Rh-SrTiO₃ powder prepared thermally showed higher turnover numbers for H₂ photoproduction and may be acting catalytically. However, little O₂ was detected with this powder. When the turnover numbers for the different titanate powders were expressed with respect to the number of surface monolayer hydroxyl groups calculated from the surface area of the powders, some turnover numbers greater than one were obtained.     

Oxygen Uptake in the Vitamin B2-sensitized Photo-oxidation of Tyrosine and Tryptophan in the Presence of Uracil: Kinetics and Mechanism

Considering the significance of visible light-promoted reactions in complex biological media, the photo-oxidation of the amino acids (AAs) tyrosine (tyr) and tryptophan (trp) was studied in the presence of the naturally occurring oxidative scavenger uracil (ur). The involved photoprocesses, studied at pH 7 and 9, are driven through the reactive oxygen species (ROS) singlet molecular oxygen (O₂(¹Δ_g)), superoxide radical anion (O₂^•−) and hydrogen peroxide (H₂O₂). The effect on the effectiveness of the overall photo-oxidation process due to the presence of an added electron-donating substrate such as ur is not straightforwardly predictable. The addition of the pyrimidine compound, a much lesser photo-oxidizable substrate than the AAs themselves, produced different results: (1) antioxidative for tyr at pH 9, decreasing the overall rate of oxygen uptake; (2) synergistic for tyr at pH 7, increasing the oxidation rate more than the corresponding addition value of the respective individual rates and (3) no effect for trp at both pH values. The final result depends on the respective abilities of the substrates as quenchers of both the long-lived riboflavin triplet excited state and the generated ROS and the pH of the medium. An interpretation for the different cases is attempted through a kinetic and mechanistic analysis.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

NEAR-UV-INDUCED BREAKS IN PHAGE DNA: SENSITIZATION BY HYDROGEN PEROXIDE (A TRYPTOPHAN PHOTOPRODUCT)

Abstract— Near-UV irradiation of l -tryptophan yields a large number of photoproducts. When this mixture is added to recombinationless ( rec ) mutants of bacteria, the cells are killed. The most toxic component of tryptophan photoproducts has been identified as hydrogen peroxide (H₂O₂). We now report that both tryptophan photoproducts and H₂O₂ sensitize phage DNA to near-UV radiation resulting in enhanced killing as well as enhanced DNA breakages. We conclude that the in situ production of H₂O₂ via tryptophan photolysis may be an important biological event.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

PHOTOLYSIS OF AQUEOUS SOLUTIONS OF POTASSIUM CIS-DIAQUOBIS (OXALATO) CHROMATE (III)

Abstract— The photolysis of aqueous solutions of cis -[Cr(C₂O₄)₂(H₂O)₂]- at 254 nm and pH 4 produced CO₂ and H₂ in nearly equal yields. The quantum yield of hydrogen, φ², increased by 9% and the yield of carbon dioxide, φ, by 65% as the pH was lowered from 4 to I. The total gas yield, φ_gas, decreased in the presence of added oxalate or chromium (II) ions and when the light intensity was lowered. The gas yield in D₂O was appreciably higher than in H₂O. The variation of φ_gas with pH (D) and with added oxalate ion was roughly parallel in the two liquid media. The gas yield increased in the series:
A tentative mechanism is suggested to explain the results.     

THE HABER-WEISS CYCLE

Abstract— The Haber-Weiss cycle:
was investigated at low pH by radiolysis of oxygen or nitrogen saturated solutions of hydrogen peroxide. It was found that reaction 2 has a low rate constant: k ₂= 3.0 ± 0.6 M ^-1 s^-1 (pH 2.3, 22°C). The rate determining step of reaction 2 is most probably the transfer of an electron from a π₈* orbital of HO₂ to the empty _u* orbital of H₂O₂. Overlap between these two orbitals is hindered by the filled π₈* orbitals of H₂O₂. Fe(HI)EDTA catalyses reaction 2.     

CATALYTIC ACTION AND DESTRUCTION OF PROTOHEMATIN DURING PEROXIDE DEPENDENT LUMINOL CHEMILUMINESCENCE

Abstract— The catalytic action of protohematin was studied during the H₂O₂-dependent chemiluminescent luminol reaction. In spite of the fact that the catalyst was ultimately inactivated, the average protohematin molecule catalyzed the consumption of about 10³ molecules of luminol. The inactivation of catalyst and the initial consumption of luminol were studied during the luminescent reaction with different concentrations of reactants. A scheme accounting for the experimental observations is proposed. The formation of a primary protohematin-H₂O₂ complex is followed by binding of luminol, resulting in a ternary complex. A nucleophilic attack by a second molecule of H₂O₂ on the luminol molecule results in light emission from excited aminophthalate via a hypothetical peroxide adduct. The destruction of protohematin occurs via the attack of H₂O₂ on the porphyrin structure of the protohematin-H₂O₂ complex. Second order rate constants for the destruction of protohematin, the formation of the luminol complex and the nucleophilic attack of H₂O₂ are presented.     

Near-infrared Light Emission from Nanomolar-level Singlet Molecular Oxygen Generated with an Ultra low Concentration Mixture of Hypochlorite and Hydrogen Peroxide

Abstract— We report the detection of a weak near-infrared light emission originating from 8 nM singlet molecular oxygen (¹O₂) produced in a mixture of 1 m M hypochlorite (OC1^-) and 8 n M hydrogen peroxide (H₂O₂). The measurements were made with a highly sensitive detection system for ultraweak light emission in the 1.0-1.5 μm wavelength region. The emission intensity exhibited linear dependence for H₂O₂ concentrations in the range of 8-670 n M . The mixture containing a lower concentration (33 μ M ) of OCl^- pseudocontinuously emitted near-infrared light for 5 s. The rate constant for ¹O₂ production obtained from the kinetic analysis agrees with that previously reported. Our results demonstrate the possibility of measuring very low concentrations of ¹O₂ in a OCi-/H₂O² mixture as well as ¹O₂ production in intact living systems.     

Increased UV-B Radiation Affects the Viability, Reactive Oxygen Species Accumulation and Antioxidant Enzyme Activities in Maize (Zea mays L.) Pollen

The increase in UV-B radiation reaching the earth's surface has prompted extensive studies on the effects of UV-B on plants. However, most of these studies have not addressed the close characteristics related to future survival of plant populations. The purpose of this study was to investigate the effects of UV-B radiation on reactive oxygen species (ROS) accumulation and antioxidant defense system in relation to germination, tube length and viability of maize pollen. Our results indicate that increased UV-B radiation decreased the pollen germination rate and tube length in vitro and also its fertilization ability in the field. Production of O₂^•− and H₂O₂ increased by UV-B radiation treatment, and their continuous accumulation resulted in lipid peroxidization. The activities of superoxide dismutase, catalase, peroxidase and DPPH-radical scavenging were decreased by increased UV-B radiation. The increased ROS and lipid peroxidization, and decreased activities of the antioxidants may be attributed to the effects of UV-B radiation on pollen germination, tube growth and fertilization ability.