INFLUENCE OF pH AND OXYGEN ON 315 mμ INACTIVATION AND THYMINE DIMERIZATION IN MUTANTS OF BACTERIOPHAGE T4

Abstract— 1. Irradiation with 315 mμ light inactivates phage T4v-x C, and T4v^-x- , and forms thymine dimers in their DNA.
2. Both the rates of inactivation and of thymine dimerization depend upon pH and gaseous environment during irradiation. The U.V. sensitivities are: 1 (pH 7, N₂, 03, 2.2 (pH 3.5, Oz), 3.3 (pH 3–5, N₂; and the corresponding rates of thymine dimerization 1: 2.5: 5.2. The number of thymine dimers per lethal hit observed withT4v-x + are: 5.7 (pH 7, N₂, O₂, 5.4 (pH 3.5, O₂, 10.9 (pH 3.5, N₂); and forT4v-x-: 4.6, 3.4, and 7.1 with the same sequence of conditions.
3. Also the photoreactivable sectors depend upon the environmental conditions at 315 mp inactivation. In T4v-x f this sector amounts to about 50 per cent at pH 7, 18 per cent at pH 3.5, O., and 29 per cent at pH 3.5, N, respectively.
4. The molecular basis of these findings is discussed. It is concluded that, besides thymine dimer, at least one other lethal photoproduct (probably a photoproduct of cytosine) is involved in photoreactivation.     

PHOTODYNAMIC EFFECTS OF PROFLAVINE ON BACTERIOPHAGE φ times 174 AND ITS ISOLATED DNA

Abstract. Proflavine-mediated photoinactivation of φ times 174 phage and its isolated DNA was studied under identical irradiation conditions. The inactivations followed single-hit kinetics and a linear relationship was obtained in reciprocal plots of the inactivation rates vs the proflavine concentrations for both phage and isolated DNA. The phage photoinactivation rate was increased with an increase in the amount of proflavine bound to the phage DNA in a strong binding range (0.01-0.04 proflavine/ nucleotide) as the total proflavine concentration was increased or the ionic strength decreased. Further, a phage-specific factor was also found to affect the inactivation rate. The photodynamic treatment induced mutations in three phage strains from "amber" to "wild type" at a mutation rate per lethal hit of 0.3 times 10^-5 to 2.6 times 10^-5. In contrast to phage infectivity, the φ times 174 DNA infectivity was measurable only at a high multiplicity of infection, and its photoinactivation occurred only at high proflavine concentrations. The photoinactivation rate was enhanced either with a decrease in the multiplicity of infection or with the use of spheroplasts of recA mutants strains. The results are discussed in terms of the nature of and possible repair mechanisms of photodynamically induced lesions in φ times 174 phage DNA.     

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.     

A PHOTOSYSTEM I-PHENOSAFRANINE SOLAR CELL

Abstract Bacteriophage λ_vir was inactivated when it was irradiated with near-UV light in the presence of chlorpromazine. DNA strand breakage in the treated phage was indicated by alkaline sucrose gradient centrifugation. The number of the breaks was increased with increasing fluence. Although the inactivation rate was enhanced with a decreasing salt concentration in the reaction mixture and under a nitrogen atmosphere, the number of the strand breaks was not altered in either case. Therefore, the DNA strand breakage is not a sole lethal damage in the treated phage. The addition of NaN₃ repressed the inactivation and the reaction in a D₂O medium enhanced the inactivation even if the reaction mixture was irradiated under anaerobic conditions. Under anaerobic conditions, the inactivation occurs presumably via a radical mechanism.     

MECHANISMS FOR DYE-MEDIATED PHOTODYNAMIC ACTION: SINGLET OXYGEN PRODUCTION, DEOXYGUANOSINE OXIDATION AND PHAGE INACTIVATING EFFICIENCIES

Abstract The production of singlet oxygen (¹O₂) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6-tetramethyl-4-piperidone as ¹O₂ acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of ¹O₂ production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of ¹O₂ was not observed with 9-aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the ¹O₂ production and (ii) the bacteriophage ØX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated ¹O₂. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

PHOTOINDUCED LUMINESCENCE AND EPR SIGNALS OF POLYPHENOL AND QUINONE POLYMERS

Abstract— Aqueous basic solutions, pH 9.0 of humic acids and melanin-like, synthetic polymers, obtained with adrenochrome, hydroquinone and purpurogallin, were illuminated with visible light under N₂ or O₂ atmospheres. It has been found that light enhances a singlet electron-paramagnetic-resonance (EPR) signal of polymers both under N₂ and O₂, and induces ultra-weak luminescence in the presence of O₂. Degradative oxidation of polymers, accelerated by light, leads to a decrease of EPR signal intensity and generates weak chemiluminescence.     

CONVENIENT AND SIMPLE METHODS FOR THE OBSERVATION OF PHOSPHORESCENCE IN FLUID SOLUTIONS. INTERNAL AND EXTERNAL HEAVY ATOM AND MICELLAR EFFECTS

Abstract— Phosphorescence of organic molecules in fluid solutions may be conveniently and readily observed under certain conditions. If k _p (radiative phosphorescence rate constant) is 10s^-1, then (in the absence of photoreaction) phosphorescence is observable upon N₂ purging. For example, nitrogen purged, acetonitrile solutions of bromo and dibromonaphthalene display readily observable phosphorescence as a result of internal heavy atom enhancement of π_S, and k _p. External heavy atom enhancement of k, (CH₂BrCH₂Br solvent) of aromatic hydrocarbons even allows observation of phosphorescence from these compounds in N₂ purged fluid solutions. Although bromonaphthalenes are not significantly phosphorescent in N₂ purged aqueous solution, phosphorescence is readily observed in N₂ purged detergent (HDTBr, HDTCl, and SDS) solutions above the critical micelle concentration. The general factors which determine whether phosphorescence is "readily" obervable in fluid solution are briefly discussed and the results are interpreted in light of these factors.     

PHOTOVOLTATC EFFICIENCIES OF MICROCRYSTALLINE AND ANHYDROUS CHLOROPHYLL a

Abstract— A thin layer of chlorophyll a (around 2000 Å). a p -type organic semiconductor. was sandwiched between two different metals. aluminum and silver. We used the photovoltaic effect in order to study the efficiency of light conversion by the crystalline and anhydrous forms of Chl a . When the photovoltaic cell is illuminated through the semi-transparent aluminum electrode. an action spectrum similar to the visible absorption spectrum of Chl a is obtained. The anhydrous form. always shows a maximum, in the red, at 672 nm and the crystalline one at 746 nm to 738 nm depending on the amount of water vapor present in the measuring area. The light conversion efficiency has been measured at the maximum absorption in the red for both forms of Chl a . For the anhydrous form, we found values as high as 0.036%, which is very significant even compared to 0.21%, the highest value found for the crystalline form. In both cases. the incident light power was approximately 10 μW on the cell. The light conversion efficiency of both forms of Chl a cells was found to depend on a combine effect of the nature of the gas (O₁. N₂. air. Ar. N₂O. SF ₆ , H₂) and the amount of water vapor present in the measuring area. The best conversion efficiency was obtained with O ₂ , or air saturated with water. This combined effect was very large for the crystalline form whereas the anhydrous form was only slightly affected.     

DETERMINATION OF SINGLET OXYGEN RATE CONSTANTS IN AQUEOUS SOLUTIONS

Abstract— A very efficient quenching of singlet oxygen (¹O₂) by N₃^- ions has been applied to the determination of rate constants of reactions of ¹O₂ with various substrates (A). This determination has been made possible by choosing experimental conditions which give simple competition between N₃^- and A for ¹O₂ formed in the steady state irradiation of convenient sensitizing dye (S). The consumption of oxygen by the substrate, as followed with an oxygen analyzer, decreases in the presence of low concentrations of N₃^-. Using neutral air saturated aqueous solutions containing the dye phenosafranine + A and varying concentrations of N₃^-, the ¹O₂ rate constants for reactions with biological substrates and some radiation protective agents have been determined.     

SINGLET OXYGEN: A MAJOR REACTIVE SPECIES IN THE FUROCOUMARIN PHOTOSENSITIZED INACTIVATION OF E. COLI RIBOSOMES

Abstract— The skin photosensitizing furocoumarins, 8-methoxypsoralen (MOP) and 4,5',8-trimethylpsoralen (TMP), inactivate E. coli ribosomes in vitro , on UV irradiation at 313 nm. Purging the solutions with N₂ protects the ribosomes considerably against photoinactivation (75% with MOP and 80% with TMP). In air, the ribosome photoinactivation is mainly due to singlet oxygen (¹O₂), since the presence of NaN₃ and other ¹O₂ quenchers protects the system and the inactivation is enhanced in D₂O. Although ¹O₂ dominates as the inactivating species, the possibility of additional (∼15%) minor mechanisms involving free radicals exists. However, O^-₂ does not appear to be the damaging species, since superoxide dismutase does not provide any protection.
Photosensitization of the partially purified enzyme, phe-tRNA-synthetase with MOP or TMP shows inactivation and protection curves similar to those seen with the ribosomes. On the other hand, unfrac-tionated tRNA^phc is not photosensitized under similar conditions, although it shows self-photosensitization. It is likely that in the furocoumarin-sensitized ribosomes, the primary events of photoinactivation are associated with the proteins.     

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.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

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.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.     

EFFECT OF SODIUM AZIDE ON PHOTODYNAMIC INDUCTION OF GENETIC CHANGES IN YEAST

Abstract— In view of the increasing attention to ¹O₂ (¹Δ_g) participation in the photodynamic action, different types of genetic changes in Saccharomyces cerevisiae by acridine orange sensitization were compared with respect to the response to N₃^-, a well known quencher of ¹O₂. The induction of mitotic crossing over with respect to ade 2 locus and mitotic gene conversion at trp 5 locus were suppressed by the addition of N₃^- suggesting the involvement of ¹O₂ as a major intermediate. However, the induction of reverse mutation at ilv 1 was only slightly suppressed. These results may indicate that there are two types of photodynamic DNA damage; one is produced via ¹O₂ and the other via non-¹O₂ reaction pathway which lead to mitotic gene conversion and mitotic crossing over, and to mutation, respectively.     

ALTERATION OF GUANINE RESIDUES DURING PROFLAVINE MEDIATED PHOTOSENSITIZATION OF DNA

Abstract— Mild photodynamic treatments of proflavine-calf thymus DNA complexes induce a unique and quantitatively important alteration of the guanine residues which can be related to the lethal lesions due to the combined action of proflavine and light on phages. The 'altered guanine' is destroyed by HClO₄ but is recovered after partial DNA depurination under the form of two photoproducts. The first product, G_ox, elutes as guanine on a Sephadex column but has a modified UV absorbance spectrum. It gives rise by further irradiation to another product, X, which elutes at pH 9.7 as a pyrimidine compound and presented a maximal UV absorbance at 246 nm. Product X is also selectively released by piperidine fixation onto the photo-damaged DNA. The guanine degradation process is markedly decreased in the presence of the singlet oxygen quencher, NaN₃. The photodynamic lesion inhibits the enzymatic degradation of the DNA but generates locally denatured regions that are sensitive to S1 endonuclease.     

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.     

USE OF POTASSIUM IODIDE AS A CHEMICAL ACTINOMETER

Abstract Aqueous solutions of KI were examined for use as chemical actinometers to measure 254 nm (germicidal) radiation. Irradiation results in electron ejection from iodide such that aqueous electrons and iodine atoms are formed. In the presence of N₂0, an electron scavenger, recombination of these two reactive species is eliminated and stoi-chiometric formation of triiodide occurs. The absorbance increase due to triiodide was followed and the quantum yield determined using either a radiometer or ferrioxalate actinometry to estimate the amount of energy absorbed by the KI solution. The quantum yield ( ø ) at 25°C was determined to be 0.26 using radiometry, and 0.224–0.233 using actinometry, depending on the radiation conditions. The following expression was used to measure the incident Huence rate for 254 radiation at a given temperature T for irradiation over a time interval Δt (s) fluence rate (W/m²) = 4. 96 × 10⁶ΔOD(λ)/ e(λ) [0.23+0.004(T-25)] δ t cm⁻¹
At temperatures other than 25°C, the temperature dependence of the quantum yield (0.004/°C) is taken into account by the term shown in the denominator. Because KI remains relatively blind to longer wavelengths such as those found in normal room light, measurements can be made in the presence of room light without having to work in a darkened room.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.