INACTIVATION OF LIPID-CONTAINING VIRUSES BY HYDROPHOBIC PHOTOSENSITIZERS AND NEAR-ULTRAVIOLET RADIATION

Abstract—The hydrophobic photosensitizers acridine and phenothiazine inactivate the lipid-contnining viruses PM2,φ6, and herpes simplex when samples are illuminated with near-UV radiation. φ23–1- a . which is insensitive to organic solvents and presumably contains no lipids. is not inactivated under comparable conditions. For acridinc, the inactivation of virus requires that oxygen be present and is inhibited by sodium azide, implicating the involvement of singlet oxygen. For phenothiazine, oxygen is not required for photosensitized inactivation. Treatment of PM2 with acridine and near-UV light caused a complete disruption of the virion, as determined by sucrose gradient analysis of treated and untreated samples. These data and related observations suggest that lipid-containing viruses are inactivated through photosensitized membrane damage.     

MEMBRANE DAMAGE CAN BE A SIGNIFICANT FACTOR IN THE INACTIVATION OF ESCHERICHIA COLI BY NEAR-ULTRAVIOLET RADIATION

Abstract A DNA repair competent strain of Escherichia coli K-12 showed sensitivity to inorganic salts (at concentrations routinely used in minimal media) after irradiation with broad spectrum near–UV radiation, at fluences that caused little inactivation when plated on complex growth medium. This effect was not observed with cells that had been exposed to 254 nm radiation. This sensitivity to minimal medium was increased by increasing the salt concentration of the medium and by increasing the pH of the medium. This sensitivity was greatly increased by adding to the medium a low concentration of commercial glassware cleaning detergent that had no effect on unirradiated cells or far-UV irradiated cells. These findings may explain the large variability often observed in near-UV radiation survival data, and demonstrate that, at least on minimal medium plates, membrane damage contributes significantly towards cell killing. This phenomenon is largely oxygen dependent.     

REPAIR OF 313-NM INDUCED LESIONS AND PHOTOPROTECTION IN YEAST CANDIDA GUILLIERMONDII

Abstract. The present communication is concerned with the effects of near-UV radiation (300–380 nm) on yeast Candida guilliermondii. It was found that certain doses of 313 nm irradiation caused inactivation of the yeast which was exhibited in a way different from the lethal action of far-UV radiation. It was also found that the cells inactivated by 313 nm are capable of recovering vitality, if incubated for some time in a non-nutrient medium. The yeast inactivated by far-UV radiation also proved to be capable of recovering, though to a lesser degree. Both 334 nm radiation and non-lethal doses at 313 nm induced the photoprotective effect against far-UV damage. The effect was exhibited if there was a certain time interval (2–4 h) between the exposures to photoprotective light and subsequent far-UV radiation. Within this time interval the extent of photoprotection was dependent on temperature.     

LIPID PEROXIDATION AND OTHER MEMBRANE DAMAGE PRODUCED IN Escherichia coli K1060 BY NEAR-UV RADIATION AND DEUTERIUM OXIDE

Abstract— With accumulating evidence that the membrane is an important site for near-UV inactivating events, we have investigated the effects of near-UV radiation on the unsaturated fatty acid auxotroph E. coli K1060 following incorporation into the membrane phospholipids of fatty acids with varying degrees of unsaturation. Sensitivity, lipid peroxidation and membrane damage, as determined by ⁸⁶Rb⁺ leakage, have been found to increase with increasing unsaturation in log-phase cells. Similar experiments carried out in D₂O also show an increase in sensitivity, lipid peroxidation and membrane damage, indicating that singlet oxygen may be responsible for such near-UV-radiation-induced damage     

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.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

PHOTOMIMETIC EFFECT OF SEROTONIN ON YEAST CELLS IRRADIATED BY FAR-UV RADIATION

Abstract— The effect of serotonin on the survival of far-UV irradiated cells of the yeast Candida guillier-mondii was studied. Serotonin was found to have a photomimetic property. Preincubation of cells with serotonin results in protection against far-UV inactivation, whereas the post-radiation treatment with serotonin causes a potentiation of far-UV lethality. Both effects are similar to those produced by near-UV (334 nm) radiation. The observations provide support to the idea advanced by us previously that photosynthesized serotonin is the underlying cause of the two effects of near-UV radiation, photo-protection and potentiation of far-UV lethality. Experiments with an excision-deficient strain of the yeast Saccharomyces cerevisiae suggest that the effect of serotonin is by its binding to DNA.     

EARLY EFFECTS OF NEAR-ULTRAVIOLET RADIATION ON YEAST CELLS: CHANGES IN CELL SIZE

Exponentially growing yeast cells showed a drastic shrinkage when irradiated in water suspension (but not in buffer) with broad-band near-UV radiation. This was clearly seen soon after irradiation in size-distribution curves measured by a Coulter counter. This early shrinkage was observed at a near-UV fluence where the survival enters a region of exponential decline after a large shoulder. We further observed the formation of a distinct band in a density-gradient-centrifugation profile, presumably corresponding to the near-UV-affected cells. A survival test showed that the cells making up this band were dead.     

REC A +-DEPENDENT SYNERGISM BETWEEN 365 NM AND IONIZING RADIATION IN LOG-PHASE ESCHERICHIA COLI: A MODEL FOR OXYGEN-DEPENDENT NEAR-UV INACTIVATION BY DISRUPTION OF DNA REPAIR

Abstract —The oxygen dependence of 365 nm inactivation of colony-forming ability of Escherichia coli has been investigated in two series of DNA repair-deficient K12 mutants grown to mid-exponential phase. All strains except a uvr A rec A double mutant are more sensitive to inactivation under O₂ and show a lower threshold dose. The inactivation of photoreactivating enzyme in a crude cell extract and DNA repair disruption are both reduced when irradiation is carried out under nitrogen. The rec A gene-dependent synergism between 365 nm and ionising radiation is reversible if cells are incubated in full growth medium before ionising radiation treatment. In a wildtype strain, incubation for 2.5 h in full growth medium after 10⁶ J m^-2 365 nm radiation changes a sensitised response to a protection from ionising radiation. Protection is not seen at 1.5 times 10⁶ J m^-2. A tentative model for near UV lethality in logarithmic phase cells is suggested which proposes two classes of lesions. One requires oxygen for it's induction, is rapidly fixed as a lethal event as a result of repair disruption, and is primarily responsible for cell death after aerobic 365 nm irradiation. The other lesion, possibly pyrimidine dimers, may lead to cell death under anaerobic conditions.     

INACTIVATION OF NORMAL AND MUTANT NEUROSPORA CRASSA CONIDIA BY VISIBLE LIGHT AND NEAR-UV: ROLE OF 1O2, CAROTENOID COMPOSITION AND SENSITIZER LOCATION

Abstract— Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-UV light has been investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV is virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, ¹O₂ is not involved in any substantial way in the formation of lethal lesions. The finding that carotenoid deficient strains are similar to wild-type strains in sensitivity to near-UV inactivation is consistent with ¹O₂ not being involved.
Photodynamic inactivation of conidia by visible light occurs in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains are more sensitive to such inactivation only when MB and TB are used. These results support the contention that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids.
A newly isolated, lemon–yellow mutant, mapping to the al -1 locus, exhibits sensitivities to photodynamic inactivation similar to other pure-white mutants at the same locus. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation (seven to nine double bonds) of the two colored carotenoids, zeta–carotene and neurosporene, produced by the mutant.     

SIMILAR SPECTRA FOR THE INACTIVATION BY MONOCHROMATIC LIGHT OF TWO DISTINCT LEUCINE TRANSPORT SYSTEMS IN ESCHERICHIA COLI

Abstract—Kinetics of inactivation of two separate leucine transport systems (leucine specific and LIV) in E. coli by seven wavelengths of monochromatic light have been studied. Loss of leucine uptake is not due to generalized membrane damage causing non-specific leucine leakage. Inactivations are usually exponential but some wavelengths show shoulders at low doses. Two-component spectra between 254 and 435 nm occur for both transport systems. Inactivation is most efficient at 290 nm and a second peak occurs at 365 nm. Both leucine transport systems are inactivated similarly.     

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.     

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.     

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.     

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.     

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.     

AN ACTION SPECTRUM FOR ULTRAVIOLET RADIATION-INDUCED MEMBRANE DAMAGE IN Escherichia coli K-12

A wild-type Escherichia coli K-12 strain was irradiated using monochromatic radiation in the range 254 to 405 nm. A measure of the cell membrane damage induced at each wavelength was investigated by comparing cell viability after irradiation on nutrient agar and on minimal medium containing either a low or high inorganic salt concentration. An action spectrum for lethality and for cell membrane damage was then determined. From 254 to 310 nm lethality closely corresponded to the absorption spectrum of DNA, and there was no indication of membrane damage. However, above a wavelength of 310 nm, the direct absorption of radiation by DNA could not account for the sensitivity observed. Moreover, at wavelengths longer than 310 nm, cell membrane damage was induced and by an increasing factor up to a peak at 334 nm. At the longer wavelengths of 365 and 405 nm, there was a gradual decrease from the peak of damage to cell membranes induced by 334 nm radiation. These results indicate that cell membrane damage may contribute significantly to near-UV radiation-induced cell lethality in wild-type E. coli K-12.     

Structural and Functional Changes in Catalase Induced by Near-UV Radiation

Part one of this study shows that exposure of purified beef liver catalase in buffered solutions to BL lamps that provide a mixture of 99% UVA and 1% UVB (to be labeled UV_A) alters its chemistry and enzymatic activity. Thus, its spectral absorbance lost detail, it aggregated and exhibited a lower isoelectric point and its enzymatic activity was substantially reduced. These photochemically induced changes were increased by irradiation in phosphate buffer or in physiological medium (minimal essential medium) containing riboflavin and tryptophan. Neither α-tocopherol nor de-feroxamine were protective against these UV_A-induced changes in pure catalase. We further investigated the effect of UV_A radiation on the activity of catalase in cultured lens epithelial cells and the protective effects of antioxidants. Cultured lens epithelial cells of rabbits and squirrels were exposed to near-UV radiation with representation in the UVA region of 99% and 1% UVB. Catalase assays were done on ho-mogenate supernatants of cells kept dark or UV exposed. In some instances, cells were cultured in medium containing a-tocopherol or deferoxamine prior to UV radiation. Comparisons were made between UV-exposed lens cell catalase activity when exposure was done with or without the antioxidants. The UV_A radiation was strongly inhibitory to both rabbit and squirrel lens epithelial cell catalase activities. The range of fluxes of near UV radiation was compatible with that which could reach the lens from the sunlit environment. Catalase inactivation was lessened in cells preincubated with a-tocopherol and deferoxamine. This suggests that both singlet oxygen and hydroxyl radical formation may be involved in near-UV damage to lens epithelial cell catalase. Such inhibition of catalase by near-UV would enhance H₂O₂ toxicity and stimulate SH oxidation so as to damage the lens.     

TOLUIDINE BLUE: THE MODE OF PHOTODYNAMIC ACTION IN YEAST CELLS

Abstract— Toluidine blue, a thiazine dye, was shown to have in vivo photodynamic activity through singlet oxygen (O₂¹Δ _g ) production. This was based mainly on the effective protection by N^-₃ and the marked enhancement in D₂O for the sensitized inactivation of yeast cells. The mode of the in vivo activity was, however, quite different from that of acridine orange, for which the singlet oxygen mechanism has also been proposed. The most characteristic feature in the toluidine blue-sensitization was the total lack of the induction of gene conversion (at trp 5), while the survival went down below 10%. The non-induction of genetic changes was confirmed at several pH's in the neutral region, whereas the inactivation was seen in parallel to the reported pH dependence of singlet oxygen production in vitro . Direct measurements by microspectrophotometry showed none of the toluidine blue was accumulated in the cell. It was also ascertained from acridine-sensitized induction of gene conversion that toluidine blue never interfered with the binding of acridine orange to cellular DNA. These findings suggested that the unique mode of photodynamic activity of toluidine blue is attributable to its action from outside of the cell. Furthermore, comparisons between the photodynamically treated cells (with toluidine blue) and non-treated cells with respect to the response to UV irradiation excluded certain cell functions relating to the expression of gene conversion from the possible damage sites. The photo-reactivation process of UV induced gene conversion was not disturbed by the pre-toluidine blue sensitition. In view of the foregoing results, the plasma membrane was tentatively suggested as the most likely site of damage.     

MUTAGENICITY OF MONOADDUCTS AND CROSS-LINKS INDUCED IN ASPERGILLUS NIDULANS BY 8-METHOXYPSORALEN PLUS 365 NM RADIATION

Abstract— 8-Methoxypsoralen plus 365 nm radiation induces mutation at the methionine supressor loci of Aspergillus inhibitor-deficient conidia at low doses of near-UV radiation with one-hit kinetics and at higher near-UV radiation doses with two-hit kinetics. These results and others suggest that both monoadducts and cross-links, formed by 8-methoxypsoralen and DNA upon exposure to UV radiation, are capable of inducing mutation. Evidence is also presented that induced furocoumarin cross-links are responsible for the inactivation of the Aspergillus conidium.