IN SITU HYDROGEN PEROXIDE PRODUCTION MAY ACCOUNT FOR A PORTION OF NUV (300–400 nm) INACTIVATION OF STATIONARY PHASE Escherichia coli

Abstract— Pre-irradiation of stationary phase cells of Escherichia coli K-12 with broadband near-UV radiation potentiates the lethal effects of subsequent exposure to near-UV radiation plus hydrogen peroxide. Identical fluences failed to modulate killing due to far-UV radiation. These data indicate that biologically revelant levels of hydrogen peroxide may be generated in situ upon the near-UV irradiation of cells.     

DIFFERENTIAL, LETHAL AND MUTAGENIC ACTION OF 254 nm AND 320–400 nm RADIATION ON SEMI-DRIED BACTERIA

Abstract— The effect of culture conditions on the lethal and mutagenic action of 254 nm (u.v.) and 320–400 nm (b.l.) light has been examined. Ten strains of Escherichin coli were used in these investigations. It was found that semi-dehydration in aerosols greatly enhanced the lethal and mutagenic actions of both U.V. and b.l., Mutations induced by U.V. were found to be of a random kind, while those produced by b.l. were specific and of a particular biochemical type depending on the strain of cell and its stage of development. The presence of oxygen during irradiation enhanced b.l. effects but had no effect on U.V. damage while anaerobic growth endowed the cells with added resistance to b.l. and u.v., Stationary phase cells of E. coli B/r were found to be mutated by b.l. specifically at a thymine locus and to be more sensitive than E. coli B to the inhibition by b.l. of respiration. Some mutations induced by b.l. in E. coli B/r were found to hinder the cells ability to carry out the photoreversal of U.V. damage. It is suggested that b.1. affects a specific piece of DNA which is in contact with the cytochrome chain of the cytoplasmic membrane and that this contact point between the cytochrome chain and DNA alters sequentially as the cell proceeds through its life cycle.     

ULTRAVIOLET ACTION SPECTRA FOR AEROBIC AND ANAEROBIC INACTIVATION OF Escherichia coli STRAINS SPECIFICALLY SENSITIVE AND RESISTANT TO NEAR ULTRAVIOLET RADIATIONS

Abstract— Action spectra for the lethal effects of ultraviolet light (254–434 nm) irradiation delivered under aerobic or anaerobic conditions to Escherichia coli RT2 (specifically sensitive to near-UV radiation; > 320 nm) and E. coli RT4 (near-UV resistant) were prepared. Negligible oxygen dependence was observed for both strains below about 315 nm. The oxygen enhancement ratio (OER) for RT4 increased above this wavelength to the longest wavelength used, whereas for RT2 there was a greater increase in the OER to a large peak at 365 nm, then a progressive decrease at longer wavelengths. The results are consistent with the possibility that the sensitivity of strain RT2 to near-UV radiation may be due to hyperproduction of photosensitizer, operating via photodynamic type reactions involving excited species of oxygen.     

MUTATIONAL INTERACTIONS BETWEEN NEAR-UV RADIATION AND DNA DAMAGING AGENTS IN Escherichia coli: THE ROLE OF NEAR-UV-INDUCED MODIFICATIONS IN GROWTH AND MACROMOLECULAR SYNTHESIS

Abstract— The mutational interactions between near-ultraviolet (near-UV, 334 nm, 365 nm) radiation and DNA damaging agents (far-UV (254 nm) and ethyl-methanesulphonate (EMS)) were studied in strains of Escherichia coli B/r trp thy with different susceptibilities to near-UV-induced growth delay (wild-type, rel and srd ). Far-UV induced reversion to tryptophan independence is reduced while forward mutation to streptomycin is enhanced by prior exposure of the rel+ srd+ strains to near-UV radiation. The observed interactions are reduced ( rel ) or absent ( srd ) in the two mutant strains as are the corresponding growth and macromolecular synthesis delays normally observed after near-UV treatment. Quantitatively, the degree of interaction induced by near-UV pre-treatment correlates closely with the degree of protein synthesis inhibition. We propose a mechanism for the contrasting interactions at the two genetic loci based on the different pathways by which pre-mutagenic lesions may be processed. The primary chromophore for the mutational interactions would appear to be 4-thiouracil-containing transfer RNA.     

MUTATION INDUCTION BY AND MUTATIONAL INTERACTION BETWEEN MONOCHROMATIC WAVELENGTH RADIATIONS IN THE NEAR-ULTRAVIOLET AND VISIBLE RANGES

Abstract— The induction of mutations (reversion to tryptophan independence) by various UV (254, 313, 334 and 365 nm) and visible (405 and 434 nm) wavelengths was measured in exponential phase populations of Escherichia coli B/r thy trp and B/r thy trp uvrA by assay of irradiated populations on semi-enriched media. No mutations were induced in the repair proficient strain at wavelengths longer than 313 nm. Mutations were induced in the excisionless strain at wavelengths as long as 405 nm but less than expected from the known amount of DNA damage induced. Irradiation at the longer wavelengths (434, 405, 365 and 334 nm) suppressed the appearance of 254- or 313-nm-induced mutations in the repair competent strain but not in the excision deficient strain. The relative dose-requirement for mutation suppression was related to the relative efficiency of these wavelengths in inducing growth delay. These results suggest that the growth delay induced by near-UV and visible wavelengths allows more time for the 'error-free" excision repair process to act on the potentially mutagenic lesions induced by 254- and 313-nm radiations, thereby reducing the mutation frequency observed in the repair-proficient strain. The level of near-UV mutation induced in the excision deficient strain is lower than expected from the DNA damage known to be induced. It is possible that near-UV radiation induces a class of lethal lesions that are not susceptible to error-prone repair.     

CHLORPROMAZINE PHOTOSENSITIZATION–II. FAILURE TO DETECT ANY EVIDENCE FOR INVOLVEMENT OF DNA DAMAGE IN THE PHOTODYNAMIC KILLING OF ESCHERICHIA COLI IN THE PRESENCE OF CHLORPROMAZINE

Abstract— When a suspension of Escherichia coli was irradiated with near-UV light in the presence of chlorpromazine (at a concentration below a cytotoxic level), the cells were killed. Efficiency of the photodynamic killing was not influenced by the deficiency of the uvrA gene or the recA gene. Neither phenotypic reversion of E. coli Hs30R (arginine auxotroph) nor induction of lambda prophage in lysogenic bacteria was detected after this treatment.     

IN VIVO INDUCTION OF 4-THIOURIDINE-CYTIDINE ADDUCTS IN tRNA OF E. COLI B/r BY NEAR-ULTRAVIOLET RADIATION*

Abstract— Near-ultraviolet (near-UV; 320–405 nm) irradiation of Escherichia coli B/r induces the formation in vivo of ⁴Srd-Cyd adducts in transfer RNA, as evidenced by (1) fluorescence spectrum changes of tRNA extracted from irradiated cells and reduced with NaBH₄, (2) thin-layer chromatography on cellulose of hydrolysates of trichloroacetic acid-precipitable extracts of irradiated cells, and (3) comparison of these findings with adduct formation induced by near-UV irradiation of purified mixed tRNA from E. coli. The kinetics of induction of the ⁴Srd-Cyd adduct in vivo, and the near-UV fluences required, provide strong support for our earlier hypothesis that formation of these adducts is responsible for near-UV-induced growth delay in E. coli.     

RECOVERY FROM DAMAGE INDUCED BY ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT IN ESCHERICHIA COLI

Abstract— Escherichia coli cells treated with sublethal doses of acridine plus near-UV light exhibit an effective split-dose recovery response that requires an incubation period of about 30–45 min. Studies of the metabolic requirements for split-dose recovery revealed the following: (a) DNA synthesis is not required for split-dose recovery; (b) inhibition of electron transport or protein synthesis reduces the efficiency of split-dose recovery by about one-half; (c) inhibition of phospholipid synthesis or cell wall synthesis completely eliminates the split-dose recovery response. These results suggest an involvement of membrane repair mechanisms in response to damage by acridine plus near-UV light. Additional evidence for such a process was provided by more direct assays for membrane recovery. It was found that cells treated with sublethal doses of acridine plus near-UV light are sensitive to low concentrations of detergents, and lose that sensitivity upon incubation. Likewise, treated cells are susceptible to lethal osmotic shock, but can recover from this susceptibility if incubated after treatment but prior to exposure to low osmotic conditions. Based on accumulating evidence, we propose that E. coli cells are capable of repairing membrane damage resulting from exposure to acridine plus near-UV light.     

Patterns of protein synthesis in a growth delay mutant (nuv) of Escherichia coli after treatment by near-UV radiation or hydrogen peroxide

When Escherichia coli cells are stressed by hydrogen peroxide (H2O2), synthesis of a large number of proteins is repressed, while several other proteins are induced. Since there is evidence that some lethal effects of near-UV (NUV) radiation may be directly or indirectly due to hydrogen peroxide generated by NUV light, treatment of cells with NUV radiation or H2O2 might be expected to repress and induce the same set of proteins. In this study, we compared the effects of H2O2 and NUV irradiation on patterns of protein induction and/or repression which were separate from the 4-thiouridine-dependent response using growth delay mutants (nuv). Concentrating initially on the proteins that ceased synthesis following NUV irradiation in an nuv mutant, we observed that these were not the same as those that ceased synthesis following H2O2 treatment. Inspection of two-dimensional polyacrylamide gel electrophoresis proteins indicated that NUV irradiation repressed synthesis of a different set of proteins, although there was some overlap between the two (45%). It was also observed that the new proteins which appeared after each of the two treatments were different. This suggests that the induction and/or repression of new proteins following NUV irradiation is not triggered solely via oxidative stress, although there is some overlap between the proteins that are induced or repressed following the two treatments.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.     

PHOTOBIOLOGICAL ACTIVITIES OF 5-A-LKOXYPSORALENS WITH RESPECT TO THE ACTION ON Escherichia coli

Abstract— Near-UV induced activities of a series of 5-a-lkoxypsoralen derivatives (C₁ to C₈) and 5-h-ydroxypsoralen were compared with respect to lethal, mutagenic and prophage inducing effects on E. coli . Although 5-a-lkoxypsoralen derivatives having an alkoxyl chain longer than C₂ were slightly soluble, their saturated solutions containing their insoluble portion showed the significant photolethal effect on E. coli . 5-E-thoxypsoralen was less photoactive in inducing phenotypic reversion of the arginine auxotrophy than 5-m-ethoxypsoralen but it photoinduced prophage lambda from the lysogenic bacteria as well as 5-m-ethoxypsoralen. Photomutagenesis by the derivatives of C₃ to C₈ could not be detected but ca . 10% of the lysogenic bacteria photoinduced prophage in the presence of the derivatives at the maximum induction.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

SOLAR DOSIMETRY WITH REPAIR DEFICIENT BACTERIAL SPORES: ACTION SPECTRA, PHOTOPRODUCT MEASUREMENTS AND A COMPARISON WITH OTHER BIOLOGICAL SYSTEMS

Abstract— Populations of radiation sensitive spores ( Bacillus subtilis UVSSP), vegetative bacteria ( E. coli K12-AB2480) and bacteriophage ( E. coli phage T4vx) have been considered as possible biological dosimeters to integrate DNA-absorbed solar energy incident on the Earth's surface.
Irradiation of spores of B. subtilis UVSSP with monochromatic far- and near-UV radiation and solar radiation have indicated that these radiations have a similar efficiency in inducing spore photoproducts per lethal event. Action spectra for lethality taken with the three radiation sensitive biological systems show a similar pattern in each case with a broad shoulder in the 334–365 nm wavelength region. This finding indicates a relatively high susceptibility of the DNA to chemical alteration in this wavelength range. Although less sensitive to sunlight than the other biological systems tested, the B. subtilis UVSSP spore mutant has the advantage of temperature independence of inactivation, stability between irradiation and assay and a simple, reproducible irradiation and assay procedure. Field measurements have supported the utility of this mutant as a sunlight dosimeter.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

MODIFICATION OF LETHAL INTERACTIONS BETWEEN NEAR-ULTRAVIOLET (365 nm) RADIATION AND DNA-DAMAGING AGENTS

Abstract— The modification of the lethal interaction between near-UV (365 nm) radiation and a second DNA-damaging agent by incubation between treatments in either a minimal salts medium or complete growth medium has been studied in the wild-type bacterial strain Escherichia coli K12 AB 1157. The results indicate that the lethal interaction may be separated into at least two distinct processes whose evaluation may help in classifying DNA-damaging agents in terms of the repairability of the DNA lesions induced. An observation of changes when methyl methane sulphonate is given prior to the irradiation treatment indicates that this chemical irreversibly damages repair enzymes.     

LETHAL INTERACTION BETWEEN ULTRAVIOLET-VIOLET RADIATIONS AND METHYL METHANE SULPHONATE IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI

Abstract— The lethal interaction between monochromatic radiation at various wavelengths and methyl methane sulphonate was tested in strains of Escherichia coli proficient and deficient in DNA repair. In the repair proficient wild-type strain K12 AB1157, the efficiency of sensitization to MMS as a function of dose (at 334 nm, 365 nm and 405 nm) was found to be directly correlated with the dose necessary to remove the shoulder from the survival curve at the wavelength employed. The 365 nm: MMS interaction was also observed in other repair proficient E. coli strains (W3110 and B/r) but was absent in a recA and a polA strain. Pre-treatment of AB1157 with MMS leads to a much larger interaction than pre-irradiation with 365 nm. It is concluded that dose-dependent damage to DNA repair by the near-UV radiation is involved in the interaction and possibly that MMS causes irreversible damage 10 repair enzymes.     

ROLES OF THE RELA+ GENE AND OF 4-THIOURIDINE IN NEAR-ULTRAVIOLET (334 nm) RADIATION INHIBITION OF INDUCED SYNTHESIS OF TRYPTOPHANASE IN ESCHERICHIA COLI B/r

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) is known to inhibit the induced synthesis of tryptophanase by tryptophan in Escherichia coli , showing an action spectrum similar to that for near-UV-induced growth delay. The present work shows that a rel A mutant of E. coli B/r exhibits 50% as much monochromatic near-UV (334 nm) inhibition of tryptophanase induction as the wild type, and that a mutant lacking 4-thiouridine, an unusual nucleoside in tRNA, exhibits < 10% as much inhibition of tryptophanase induction. These findings indicate that 4-thiouridine is almost the sole chromophore for this effect in E. coli B/r, but that only 50% of the effect operates by a mechanism utilizing the rel A ⁺ gene product; growth delay appears not to be primarily involved.     

EFFECTS OF GLYCEROL UPON THE BIOLOGICAL ACTIONS OF NEAR-ULTRAVIOLET LIGHT: SPECTRA AND CONCENTRATION DEPENDENCE FOR TRANSFORMING DNA AND FOR ESCHERICHIA COLI B/r

The concentration dependence for the protection of isolated transforming DNA and Escherichia coli by glycerol against 365-nm monochromatic near-ultraviolet light (UV) was measured. Glycerol protection saturates at a concentration of about 0.1 M for DNA and 1.0 M for E. coli. Action spectra for glycerol protection of transforming DNA (tryptophan and histidine markers) are similar to those obtained previously for diazobicyclo[2.2.2.˜octane (DABCO) protection, with protection reaching a maximum near 350-nm UV and decreasing rapidly at wavelengths above and below 350 nm. However, glycerol protects against near-UV about twice as efficiently as DABCO. The action spectrum for protection of E. coli by glycerol against the lethal effects of near-UV was not the same as the spectrum for DNA since glycerol sensitized the cells, but not the DNA, at wavelengths longer than about 380 nm. A possible role of hydroxyl or other radicals was supported by the observation that benzoate also protected DNA against inactivation by 334-nm UV.     

OXYGEN REQUIREMENT FOR NEAR-UV MEDIATED CYTOXICITY OF α-TERTHIENYL TO ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE

Abstract— The photosensitizing activity of α-terthienyl, a naturally occurring compound from Tagetes has been studied. Fluence-response curves for the effect of α-terthienyl and near-UV radiation on Escherichia coli have been prepared. Photosensitization of E. coli was enhanced in aerobic as compared to anaerobic conditions and exogenous superoxide dismutase had a slight protective effect. Sodium azide, a quencher of ¹O₂, protected yeast cells from photosensitization with α-terthienyl. The available evidence suggests that α-terthienyl acts as a photodynamic sensitizer in vivo.