Absence of hydrogen peroxide production by or catalase action in Rickettsia prowazeki.

Glutamic acid oxidation by Rickettsia prowazeki is not accompanied by hydrogen peroxide generation, nor is added hydrogen peroxide degraded, as measured by a manometric technique.     

Inactivation of Escherichia coli by ultraviolet light and hydrogen peroxide in a thin film contactor

Apparatus for irradiating enclosed thin liquid films with ultraviolet (u.v.) light (Λ= 253.7 nm) in combination with hydrogen peroxide was used to inactivate Escherichia coli in water. Hydrogen peroxide concentrations of 2.5, 5.0 and 10.0 g/I were used and in each case synergistic inactivation was observed. At the highest concentration, a fractional survival of 1.3 times 10^-3 was obtained after 20 min; this was decreased to 3.1 times 10^-6 by simultaneous u.v. irradiation.     

Effects of bromide upon reaction of nucleosides with hydrogen peroxide induced by ultraviolet light

When ultraviolet light was irradiated on a neutral solution of deoxynucleosides and hydrogen peroxide, concentrations of all the deoxynucleosides decreased greatly. Addition of bromide in the system suppressed the reactions of 2′-deoxycytidine, 2′-deoxythymidine, and 2′-deoxyadenosine, but not that of 2′-deoxyguanosine. Addition of hydroxyl radical scavengers suppressed the reaction. The effect of deuterium oxide, an enhancer of singlet oxygen, was small. It is reported that hydroxyl radical, generating from hydrogen peroxide by ultraviolet irradiation, can react with bromide forming bromine radical, and that bromine radical reacts with bromide forming dibromide radical anion. Our result of dose dependency of bromide suggests that dibromide radical anion is the reaction species to react only with 2′-deoxyguanosine.     

Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation.

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.     

Inhibition of DNA replication by ultraviolet light.

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10J/m2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers.     

Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions

Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is a base damage common to oxidative mutagens and the major stable radiolysis product of thymine in DNA. We assessed the mutagenic potential of thymine glycols in single-stranded bacteriophage DNA during transfection of Escherichia coli wild-type and umuC strains. cis-Thymine glycols were induced in DNA by reaction with the chemical oxidant, osmium tetroxide (OsO4); modification of thymines was quantitated by using anti-thymine glycol antibody. Inactivation of transfecting molecules showed that one lethal hit corresponded to 1.5 to 2.1 thymine glycols per phage DNA in normal cells, whereas conditions of W-reactivation (SOS induction) reversed 60 to 80% of inactivating events. Forward mutations in the lacI and lacZ' (alpha) genes of f1 and M13 hybrid phage DNAs were induced in OsO4-treated DNA in a dose-dependent manner, in both wild-type and umuC cells. Sequence analysis of hybrid phage mutants revealed that mutations occurred preferentially at cytosine sites rather than thymine sites, indicating that thymine glycols were not the principal pre-mutagenic lesions in the single-stranded DNA. A mutagenic specificity for C----T transitions was confirmed by OsO4-induced reversion of mutant lac phage. Pathways for mutagenesis at derivatives of oxidized cytosine are discussed.     

Ammonium-dependent hydrogen peroxide production by mitochondria

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.     

Alteration of DNA by hydrogen peroxide

DNA is altered by the action of hydrogen peroxide, a ubiquitous body constituent. Formation of damaged species is established by changes in four characteristics of DNA: absorption in the ultraviolet, complexing with histone, adsorption by anion exchange resin and charcoal, binding of silver cations. Damaged species of DNA might lead to impaired function of the organism. Gamma globulin by complexing with DNA is a partial inhibitor of this attack by hydrogen peroxide.     

Study on the synergetic degradation of chitosan with ultraviolet light and hydrogen peroxide

Chitosan was effectively degraded by hydrogen peroxide under irradiation with ultraviolet light. The existence of a synergetic effect on the degradation was demonstrated by means of viscometry. In addition, the optimal conditions of degradation were determined on the basis of orthogonal tests. The structure of the degraded product was characterized by Fourier-transform infrared spectra (FTIR) analysis and diffuse reflectance spectra (DRS) analysis. The mechanism of the degradation of chitosan was correlated with cleavage of the glycosidic bond.     

Hydroxyl free radical production by the reaction of N-methyl-N'-nitro-N-nitrosoguanidine with hydrogen peroxide without exposure to light.

We examined hydroxyl free radical (.OH) production in the mixture of H2O2 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without exposure to light using the electron spin resonance spin-trapping technique. When the mixtures were protected from exposure to light, .OH was formed at pH 6.5 and above; it was not formed at pH 5.0 and below, consistent with our previous report. The amount of .OH trapped depended on the concentrations of MNNG and H2O2 and the pH. Nitrite ion was also detected colorimetrically at pH 6.5 and above, but not detected at pH 5.0 and below in the mixtures without exposure to light. Moreover, its production depended on the concentrations of MNNG and H2O2 and the pH. The formation of N-methyl-N'-nitroguanidine in the mixture at pH 7.8 was confirmed by thin-layer chromatography and melting point. These results suggest that nucleophilic attack by H2O2 on the nitroso nitrogen of MNNG results in the formation of N-methyl-N'-nitroguanidine and peroxynitrous acid, which degrades homolytically to yield .OH and nitrogen dioxide, resulting in the production of nitrite ion, at pH 6.5 and above without exposure to light.     

The destruction of spores of Bacillus subtilis by the combined effects of hydrogen peroxide and ultraviolet light

Ultraviolet light irradiation of bacterial spores in the presence of hydrogen peroxide has been shown to produce synergistic kills when compared with ultraviolet light (u.v.) and hydrogen peroxide used sequentially. This use in combination has been patented for the commercial sterilization of packaging before filling with UHT-processed products. Previous results have shown that lamps producing u.v. light with a maximum output at about 254 nm were extremely effective. Results obtained using a Synchrotron radiation source to produce a narrow band of irradiation now shows that the greatest kill of spores of Bacillus subtilis in the presence of hydrogen peroxide is obtained with radiation at ˜270 nm. Such results suggest that the action of the u.v. light is not directly on the spore DNA but may be related to the production of free hydroxyl radicals from hydrogen peroxide.     

Nucleotide sequence cleavages of manganese-bleomycin induced by reductant, hydrogen peroxide and ultraviolet light. Comparison with iron- and cobalt-bleomycins

The prominent DNA breakages of bleomycin-Mn complex system were induced by reductant, hydrogen peroxide and ultraviolet light, and these three induction systems gave remarkably similar nucleotide sequence cleavage modes. The preferred DNA cleavage sites at guanine-cytosine(5'----3') and guanine-thymine(5'----3') sequences were appreciably comparable to those of the corresponding bleomycin-Fe complex systems, but not identical. In contrast, the bleomycin-Co complex system significantly degraded isolated DNA only by irradiation of ultraviolet light. The present results provide valuable information on the role of transition metals on DNA cleavage reaction of bleomycin.     

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.     

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.     

Superoxide and hydrogen peroxide production by Drosophila mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.     

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct. On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v. mutagenesis.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.