ROLE OF DYE MOLECULES REMAINING OUTSIDE THE CELL DURING PHOTODYNAMIC INACTIVATION OF ESCHERICHIA COLI IN THE PRESENCE OF ACRIFLAVINE

Abstract— Photodynamic inactivation of cells is caused by damage to the regions proximal to the cell envelope or to the DNA via a singlet oxygen mechanism. For penetrating dyes the possibility of either type of damage remains. The contribution of a penetrating dye. acriflavine. remaining outside E. coli B/r cells during irradiation. towards photodynamic inactivation was investigated. It was found that this contribution was either nil or negligible.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

LIQUID HOLDING RECOVERY OF PHOTODYNAMIC DAMAGE IN E. COLI

Escherichia coli cells lose viability when irradiated with visible light in the presence of acridine dyes. There has been some controversy about whether such photodynamic damage can be repaired. Several investigations on different E. coli strains failed to reveal any significant difference in the sensitivity to photodynamic damage between radiation-resistant E. coli B/r and radiation-sensitive E. coli B_s-1 strains (Uretz, 1964; Janovska et al. , 1970) suggesting the absence of repair. On the contrary, other investigators (Rupp, 1966; Harm, 1968) strongly predicted the existence of a dark-repair mechanism for such damage. No systematic study of liquid-holding recovery of photodynamic damage has yet been reported and knowledge of repair capacity for photodynamic damage is still incomplete. In the present communication, liquid-holding-repair capability of E. coli B/r and B_s-1 for photodynamic inactivation in the presence of acridine orange or acriflavine has been investigated. These studies show that B/r can repair photodynamic damage while B_s-1 is deficient in this ability.     

EVIDENCE AGAINST DNA AS THE TARGET FOR 334 NM-INDUCED GROWTH DELAY IN ESCHERICHIA COLI*

Abstract— Growth delay was induced with near-UV (334 nm) radiation in Escherichia coli K12 bacterial strains followed by attempts at photoreactivation (PR) of this effect at 405 nm. In the UV-sensitive strain AB2480, a small PR of the observed population growth delay occurred after 334 nm irradiation at 35°C and a much larger PR after 334 nm irradiation at 5°C. However, much of the population growth delay in this strain can be explained as being due to killing, and all or most of the observed PR pertains only to this killed fraction of the population. The true cell growth delay (i.e. that of surviving cells) thus appears to be only slightly, if at all, photoreactivable. This conclusion is supported by studies with a wild-type strain KW8, which shows growth delay at non-lethal doses; this growth delay shows no PR, regardless of the temperature during 334 nm irradiation. These findings indicate that photoreactivable lesions (cyclo-butyl pyrimidine dimers) are not an important cause of near-UV-induced growth delay. Strain AB2480 lacks known dark-repair systems for DNA damage induced by far-UV (below 300 nm) radiation, yet shows the same efficiency for 334-nm-induced growth delay as the wild type, which possesses these dark repair systems. This indicates that lesions in DNA that are dark-repairable by the systems not tunctional in AB2480are not responsible for 334-nm-induced growth delay. It is possible, however, that fragmentary repair systems in AB2480 can operate on some DNA lesion that might cause growth delay. Spontaneously decaying lesions are unlikely, since growth-delay damage decays at a very low rate in non-nutrient medium. Since most of the known types of DNA damage and repair are thus eliminated, these considerations suggest that DNA damage is not involved in near-UV-induced growth delay.     

DNA TURNOVER IN BUFFER-HELD ESCHERICHIA COLI AND ITS EFFECT ON REPAIR OF UV DAMAGE

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 10⁶ daltons DNA, each of which is at least 160 nucleotides long.
While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. We suggest that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, we propose a model to explain the limited repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery).     

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.     

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.     

COMPARISON OF SURVIVAL OF A U.V.-IRRADIATED URACIL-REQUIRING STRAIN OF ESCHERICHIA COLI B IN THE PRESENCE OF CHLORAMPHENICOL AND IN THE ABSENCE OF THE ESSENTIAL METABOLITE

Abstract— Restoration of viability of E. coli B U- after a total dose of 900 ergs/mm2 U.V. in the presence of chloramphenicol and in the absence of uracil was studied. Both treatments result in the recovery of the same fraction of the irradiated bacterial population. There is no additive effect if both treatments are applied to the same irradiated culture.     

THE ROLE OF DNA POLYMERASE I IN LIQUID HOLDING RECOVERY OF UV-IRRADIATED ESCHERICHIA COLI

Abstract. –Excision of cyclobutyl dipyrimidines from, and accumulation of strand interruptions in, DNA of different strains of E. coli K12 were determined during liquid holding recovery after UV irradiation. The extent of Pyr <> Pyr excision was the same (20–25%) for both a pol A mutant ( E. coli P3478) and its parental wild type strain ( E. coli W3110); however, single strand interruptions accumulate during liquid holding of polA cells, but not in the parental strain. In contrast, excision was greatly reduced in a mutant (KMBL 1789) which is defective in the 5'→3' exonucleolytic function of DNA polymerase I. These data suggest that excision and resynthesis during liquid holding are carried out primarily, if not entirely, by DNA polymerase I. We further conclude that excision alone is both a necessary and sufficient condition to elicit liquid holding recovery, and that this excision requires a functional polymerase I 5'→ 3' exonuclease.     

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.     

ANOXIC PHOTODAMAGE IN THE PRESENCE OF PORPHYRINS: EVIDENCE FOR THE LACK OF EFFECTS ON MITOCHONDRIAL MEMBRANES

Abstract When isolated mitochondria are irradiated in the presence of 50 μg/ml hematoporphyrin or hematoporphyrin derivative in the respiratory medium, the irradiation is at least twenty-fold less effective in impairing the Ca²⁺ pump in the absence of oxygen than in its presence. This result suggests that photosensitization by non oxygen-depending pathways has few or no effects on cell membranes, a result somewhat at variance with those observed with liposomes.     

PHOTOREACTIVATION OF ESCHERICHIA COLI Bs-3 AFTER IN ACTIVATION BY 313 nm RADIATION IN THE PRESENCE OF ACETONE

Abstract— The colony-forming ability of E. coli B_s-3 can be inactivated by light of 313 nm wavelength in an acetone-sensitized photochemical reaction. This ability can subsequently be restored quantitatively by illumination with photoreactivating light. A small fraction of the population cannot be inactivated; this is assumed to be due to a complete dark repair of the lesions, whatever the dose of radiation has been. Thus, such triplet energy-transfer experiments can successfully be applied to whole cells. Since thymine dimers are formed almost exclusively, this suggests a new way of studying these lesions in relation to the biologically observable effects.     

PHOTOREACTIVATING ENZYME FROM STREPTOMYCES GRISEUS—III. EVIDENCE FOR THE PRESENCE OF AN INTRINSIC CHROMOPHORE

Evidence is presented that DNA photoreactivating enzyme from Streptomyces griseus consists of a high molecular protein part and a low molecular chromophore which is released by denaturation. The free chromophore is highly fluorescent and has an absorption maximum at 420 nm. In native photoreactivating enzyme the chromophore fluorescence is almost completely quenched and there is an additional absorption band at 445 nm. Native photoreactivating enzyme spontaneously looses its chromophore following first order kinetics as measured by the increase of fluorescence intensity. A good correlation was found between the increase of fluorescence intensity and the decrease of biological activity, stressing the importance of the chromophore-protein bond. The presence of DNA greatly retards the spontaneous release of chromophore, and with UV-irradiated DNA the photoreactivating enzyme is almost completely stable. In five different chromatographic systems, cochromatography of biological activity and enzyme-bound chromophore was found, thus ruling out the possibility that the observed chromophore belongs to a contamination in the enzyme preparation. Photoreactivating enzyme binds very strongly to Blue-Sepharose indicating the presence of a positive charge in the polynucleotide binding site.     

THE USE OF TETRAZOLIUM SALTS TO DETERMINE SITES OF DAMAGE TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN IN INTACT CELLS FOLLOWING in vitro PHOTODYNAMIC THERAPY WITH PHOTOFRIN II

Abstract A method is described utilizing the tetrazolium salts neotetrazolium chloride (NTC), triphenyltetrazolium chloride (TTC), C,N-diphenyl-N'-4,5-dimethylthiazol-2-yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of intact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transport chain through Complex I, as evidenced by the fact that the inhibition of MTT reduction was reversible by the addition of malic acid to the culture media. A second site of dark toxicity was found to be Complex IV (cytochrome oxidase). After photoirradiation of the cells, Complex I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the addition of succinic acid, whose electrons enter the transport chain at Complex II. A second and more sensitive site of photoirradiation damage was found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoirradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contrast, TTC reduction was found to be inhibited almost immediately indicating Complex IV is extremely susceptible to photoirradiation damage. Compared to other assays of mitochondrial function requiring subcellular fractionation, the use of tetrazolium salts is simpler to perform and can be done using physiologically relevant conditions.     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract— The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants deñcient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants dencient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

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.