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.     

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.     

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.     

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.     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.     

PROTECTION OF Ustilago violacea FROM TOLUIDINE BLUE PHOTOSENSITIZATION and HYDROGEN PEROXIDE INDUCED KILLING and MITOTIC RECOMBINATION BY CAROTENES

Abstract— The accumulation of (J-carotene in the ph/ph ⁺ y diploid strain of the smut fungus Ustilago violacea was associated with reduced killing and lower levels of induced mitotic recombination compared to the β-carotene lacking ph/ph⁺ w strain in response to both incandescent photosensitization and treatment with H₂0₂. The ph/ph⁺ y strain was only slightly more resistant to killing by exogenous toluidine blue (TB) photosensitization. The ph/ph⁺ y strain exhibited significantly greater levels of survival when exposed to incandescent radiation and 1.5 μ.M TB for 15 min, as well as 3.0. 0.3, 0.03, 0.003% H₂0₂ in the dark. The ph/ph⁺ y strain also exhibited lower levels of mitotic recombination after endogenous TB photosensitization and the latter two H₂0₂ treatments. Similar survival results were obtained for the carotene accumulating haploid strain l.C2y and the carotene lacking haploid strain l.C2iv in response to H₂0₂ exposure.     

INACTIVATION OF PHAGE BY NEAR-ULTRAVIOLET RADIATION AND HYDROGEN PEROXIDE

A series of phage with different genomes (both single-stranded and double-stranded RNA and DNA) was inactivated with hydrogen peroxide (H₂O₂) in various combinations with far-ultraviolet (FUV) and near-ultraviolet (NUV) radiations. In every case but one (a lipid-coated phage), a sublethal H₂O₂ concentration greatly enhanced killing by NUV but not FUV. Moreover, this NUV/H₂O₂ synergism was oxygen independent and there was little if any host cell reactivation upon NUV plus H₂O₂ inactivation. These results suggest that these phage are inactivated by a common mechanism irrespective of nucleic acid composition, but that some phage genomes may be more vulnerable to NUV/H₂O₂ inactivation than others.     

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.     

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.     

New Results on the Photochemistry of Biopterin and Neopterin in Aqueous Solution

New photochemical studies of the reactivity of biopterin (BPT) and neopterin (NPT) in acidic (pH = 5.5) and alkaline (pH = 10.5) aqueous solutions at 350 nm and room temperature were performed. The photochemical properties of BPT are of particular interest because the photolysis of this compound takes place in the white skin patches of patients affected by vitiligo. The photochemical reactions were followed by UV/VIS spectrophotometry, HPLC, electrochemical measurement of dissolved O₂ and enzymatic methods for hydrogen peroxide (H₂O₂) and superoxide anion (O₂^•−) determinations. When BPT or NPT are exposed to UVA radiation, a red intermediate, very likely 6-formyl-5,8-dihydropterin, is generated in an O₂-independent process. That product is rapidly oxidized on admission of O₂ to yield 6-formylpterin and H₂O₂. When the photolysis takes place in aerobic conditions, no additional pathways exist. On the other hand, in the absence of O₂, the intermediate generated is not stable and leads to the formation of many products. O₂^•− is also generated during photo-oxidation of BPT and NPT. The quantum yields of reactant consumption depends on the O₂ concentration: the higher the O₂ concentration, the lower the quantum yields. This behavior is discussed in connection with the excited state of the pterins.     

ACTION OF HYDROGEN PEROXIDE ON HUMAN FIBROBLAST IN CULTURE

Abstract— Human fibroblasts in culture lose the capacity of proliferating when exposed to hydrogen peroxide in the concentration range of 1 to 10 μ M . The toxicity of H₂O₂ to xeroderma pigmentosum cells (XP12RO). defective in excision repair of lesions produced by UV-irradiation, was about twice as high as to cells proficient in excision repair (VA13). This compound produces single-strand breaks in intracellular DNA but not in purified DNA. These breaks are in situ physical discontinuities rather than alkali-labile bonds, and their generation occurs at the same extent at 4°C and 37° indicating that they are not produced by an endonuclease. The results favor the hypothesis that H₂O₂ reacts in the cell producing a radical species which brings about the formation of DNA single-strand breaks. These breaks are effectively repaired by both XP12RO and VA13 fibroblasts. The possible reason for the lethality of H₂O₂ is discussed.     

PORPHYRIN-SENSITIZED PHOTOREACTIONS IN THE PRESENCE OF ASCORBATE: OXIDATION OF CELL MEMBRANE LIPIDS AND HYDROXYL RADICAL TRAPS

Abstract— Photooxidation reactions in ascorbate (AH)-containing erythrocyte membrane suspensions have been studied in broad perspective by simultaneously monitoring lipid peroxidation in the membrane compartment and formation of hydrogen peroxide (H₂O₂) and hydroxyl radical (OH) in the aqueous compartment. Non-bound uroporphyrin (UP) and membrane-bound protoporphyrin (PP) were used as sensitizers. Photoreduction of UP to the radical anion (UP^-) was detected by electron spin resonance when UP/AH/membrane mixtures were irradiated anaerobically. Aerobic irradiation resulted in a strong AH^--stimulation of lipid peroxidation, H₂O₂ formation, and OH^- generation (detected with 2-deoxyribose (DOR) and the spin trap 5,5-dimethyl-l-pyrroline-N-oxide). Use of diagnostic agents (e.g. catalase, desferrioxamine, mannitol) revealed that OH^- is involved in light-stimulated DOR oxidation, but not in lipid peroxidation. Similar irradiation in the presence of PP resulted in far greater lipid peroxidation than observed with UP, but less DOR oxidation, and insignificant accumulation of H₂O₂. This suggests that photoreduction of membrane-bound PP is less efficient, possibly due to hindered access of AH^-.     

Sister chromatid exchanges and chromosome aberrations in human cells induced by H2O2 and other photoproducts generated in fluorescent light-exposed medium

Exposure of Dulbecco's modified Eagle's tissue culture medium to visible fluorescent light generated photoproducts toxic to human cells in culture. Toxicity manifested at the chromosome level was increased chromosome aberrations and sister chromatid exchanges in cells exposed to the photoproducts. Hydrogen peroxide (H₂O₂), a major photoproduct, induced SCE but failed to increase chromosome aberrations. Pure H₂O₂, or the H₂O₂ generated in light-exposed medium, was necessary and sufficient for inducing all the increase in SCE. However, H₂O₂ was necessary but insufficient to cause most of the chromosome aberrations. Only when acting synergistically with other photoproducts did H₂O, induce extensive chromosome aberrations. The relatively high cell densities at near confluence levels used in these experiments were less sensitive to light-induced effects, nevertheless the entire light exposure dosage range effected photoproduct production adequate for inducing SCE and chromosome aberrations. Thus, mammalian tissue and cell culture media can receive sufficient dosage from fluorescent lights illuminating rooms and culture hoods for generation of photoproducts causing gross and insidious SCE and chromosome alterations.     

A PULSE RADIOLYSIS STUDY OF THE ONE-ELECTRON OXIDATION AND REDUCTION OF 5, 10-METHENYLTETRAHYDROFOLATE

Abstract— The one-electron reduction and oxidation of 5,10-methenyltetrahydrofolate has been studied in aqueous solution in the acidity range H₀= -1 to pH = 7 using the reducing species CO^-₂ and (CH₃)₂-COH and oxidising species Br^-₂, and H₂SeO+₃. The spectral and other properties of the radicals so formed were found to be indcpendent of the reductant/oxidant used. Two protolytic forms of both the oxidised and reduced radicals were observed with approximate p K , values of 0.5 ± 0.3 being determined. Both the bridged form (5.10-methenyltetrahydrofolate) and the unbridged form (5- formyltetrahydrofolate) were found to be easily oxidised, whereas only the former could be reduced.     

PHOTOOXIDATION OF EPINEPHRINE SENSITIZED BY METHYLENE BLUE—EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN AND OF SUPEROXIDE

Abstract— The photooxidation of epinephrine, sensitized by methylene blue or by chlorophylls, excited with red light, involves the reduction of two molecules of oxygen to hydrogen peroxide per molecule of epinephrine oxidized to adrenochrome. The initial rates of these reactions are not affected by low concentrations of catalase. In 99 mol % D₂O, rates of methylene blue sensitized photooxidations are accelerated as much as 5.2 times over rates in ordinary water. Azide anion is a more effective inhibitor of this reaction in D₂O than in H₂O. Half maximal inhibitions are obtained by 1.3 mM azide in H₂O and by 0.1 mAf azide in D₂O. Isotope effects and azide sensitivities point to photooxidation of epinephrine in D₂O primarily by a singlet oxygen pathway; in H₂O, non-singlet oxygen pathways become more predominant. Superoxide dismutase (SOD) markedly inhibits rates of the photooxidations in H₂O and in D₂O; about 25% at 10^-9 M SOD, and 50% at 10^-6 M SOD in H₂O. In the photooxidation in H₂O, both by non-singlet and singlet oxygen mechanisms, the amount of superoxide produced is equivalent to the amount of O₂ consumed in the photooxidation of epinephrine; the superoxide thus formed participates in the oxidation of epinephrine.     

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.     

THE EFFECTS OF LIQUID HOLDING ON NEAR-UV(300–400 nm) IRRADIATED ESCHERICHIA COLI STRAINS DIFFERING IN NEAR AND FAR-UV RADIATION SENSITIVITY

Abstract— Stationary phase cells from four Escherichia coli strains differing in near- (nur vs. nur ₊) and far-UV (recAl vs. recA₊) radiation sensitivity were subjected to near-UV radiation (NUV) in 0.85% saline. Although the NUV-irradiated cultures yielded increased colony numbers following 24 h of liquid holding (LH), a fluctuation test for each experiment showed that the observed increases were not due to recovery but were in fact due to cell multiplication. The decline in viability observed after NUV with liquid holding using the fluctuation test was equivalent in strains RT2, 3 and 4 while the decline observed with RT1 was less marked. The discrepancy between LH involving cell densities of 10₈-10₉ and 1–4 cells/m/ can be resolved by assuming that with dense cell suspensions, NUV-induced membrane damage leads to leakage or lysis, supplying sufficient nutrients to allow growth of undamaged, surviving cells.     

Photochemical Reactions and Phototoxicity of Sterols: Novel Self-perpetuating Mechanism for Lipid Photooxidation

Abstract— Sterols are important lipid components that may contribute to phototoxicity. We have found that phototoxic response in earthworms is related to sterols extractable with lipophilic solvents. The photochemically active compounds in worm lipids are 5,7,9(11),22-ergostatetraen-3bT-ol (9-DHE) and 5,7,9(11)-cholestatrien-3bT-ol (9-DDHC), respectively. Human skin lipids are known to contain 9-DHE. We have also found 9-DDHC in human skin, which is reported here for the first time. In the presence of an excess of the corresponding 5,7-dienes (ergosterol or 7-dehydrocholesterol), these photoactive sterols constitute a self-regenerating source of singlet molecular oxygen (₁O₂) during irradiation in vivo or in vitro with UVA bT15-400 nm). The quantum yield for photosensitization of ₁O₂ by 9-DHE was estimated to be 0.09. The ₁O₂ is scavenged by the dienes and the rate constant for ₁O₂ quenching by ergosterol was found to be 1.2 times 10₇ M -₁ s_-1 in methyl t-butyl ether (MTBE). This scavenging ultimately leads to the production of 5,8-endo-peroxide and hydrogen peroxide. Photochemically induced superoxide radical was also produced on irradiation of sterol 5,7,9-trienes and trapped with the spin trap 5,5-dimeth-yl-1-pyrroline W-oxide (DMPO). The production of singlet oxygen, peroxides and radicals by the sterols may be significant in the cell damaging and tumor promoting action of UVA light on skin.     

KINETIC STUDIES OF SELF-SENSITIZED PHOTOOXYGENATION IN H2O AND D2O OF A WATER-SOLUBLE RUBRENE DERIVATIVE

Abstract A continuous argon laser has been used to study the self-sensitized photooxidation of potassium rubrene-2,3,8,9-tetracarboxylate in oxygen-saturated H₂O and D₂O. An analysis of the data obtained in concentrated solutions leads to an unexpected high value of the ratio of ¹O₂ lifetimes in D₂O and H₂O, ^T d ₂o/^T h ₂o =17 ± 1. Results obtained in diluted aqueous solutions are interpreted in terms of a re-encounter of ¹O₂ and ground state substrate molecules generated in the same triplet—triplet annihilation act.     

LIGHT-DEPENDENT AND LIGHT-INDEPENDENT PRODUCTION OF HYDROGEN GAS BY PHOTOSYNTHESIZING RHODOSPIRILLUM RUBRUM MUTANT C

Abstract. Rhodospirillum rubrum mutant C grew photosynthetically in the light and produced copious amounts of H₂. During light-growth mutant C produced 7.9mmol of H₂ in medium with 9mmol of Na-pyruvate per mg protein. When parent strain R. rubrum S ₁ was grown similarly, these cells only produced a trace amount of H₂. Light-grown mutant C evolved H₂ by H₂-nitrogenase and formic hydrogenlyase. Although both hydrogenases were previously detected in R. rubrum S₁, the activities of the reactions in light-grown mutant C were higher and they operated under different conditions. In the parent strain S₁, the production of nitrogenase was strongly repressed during growth in medium enriched with organic nitrogen and the cells only reduced 0.06 pmol of acetylene per mg protein after 30 min in the light. Under similar conditions, nitrogenase activity measured by the acetylene reduction test in mutant C was 10-fold greater. In addition to nitrogenase, mutant C also produced large amounts of H₂ with formate as an intermediate when the cells were grown with Na-pyruvate. Formic hydrogenlyase in mutant C operated equally well in anaerobic light or dark conditions. The analogous formate oxidation reaction in parent strain S₁ only functioned in the dark. These data, compared with results with R. rubrum S₁ suggested that C was a regulatory mutant. Additional observations suggested that formic hydrogenlyase occurred constitutively in R. rubrum . Pyruvate formate-lyase, however, which produced formate for formic hydrogenlyase, was only detected in the cells after growth in media with Na-pyruvate. The reaction was not formed when R. rubrum was grown in the light in media with dl-malate as the sole carbon substrate.