THE PHOTOTOXICITY OF 8-M-ETHOXYTHIONEPSORALEN AND 6-M-ETHYLTHIONECOUMARIN

The phototoxicity of 8-methoxythionepsoralen (8-MOTP) and 6-methylthione coumarin (6-MTC) when activated by UV-A has been investigated using a variety of Escherichia coli strains, Haemophilus influenzae transforming DNA and Escherichia coli pBR322 plasmid DNA. The results demonstrate that 8-MOTP is a strictly oxygen independent photosensitizer that is about 500-fold less efficient in forming lesions leading to equivalent lethality than is the parent compound from which it is derived (8-MOP). As is true for 8-MOP, 8-MOTP is capable of inducing histidine independent mutations in E. coli and inactivating transforming DNA consistent with DNA being a target for lesions induced by this molecule in the presence of UV-A. 6-MTC is a strongly oxygen dependent photosensitizer activated by UV-A when tested with either E. coli cells or transforming DNA in contrast to the parent compound (6-methylcoumarin; 6-MC) which is not phototoxic when treated with UV-A. These results imply that the membrane may be an important target leading to lethality. 6-MTC in the presence of UV-A can inactivate pBR322 plasmid and Haemophilus influenzae transforming DNA activity in vitro suggesting that DNA is a potential target for this molecule when activated by UV-A.     

EFFECTS OF ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT ON THE OUTER MEMBRANE OF ESCHERICHIA COLI

Abstract The hydrophobic photosensitizer acridine plus near-ultraviolet light damages both plasma membranes and outer membranes in Escherichia coli. Two lines of evidence are presented that outer membrane proteins are affected by acridine plus near-ultraviolet light treatment and that the effect is selective for certain proteins. First, analysis of outer membrane proteins on sodium dodecylsulfate polyacrylamide gels revealed that some protein bands are diminished upon treatment while others remain unaltered. New bands also appear, suggesting degradation or crosslinking reactions. Second, bacteriophage adsorption studies showed that treatment of E. coli F cells with acridine plus near-ultraviolet light causes a loss in functionality of the receptor for phage T5. Treatment of E. coli ABU57 cells under comparable conditions has no discernable effect on the functionality of the receptor for phage BF23.     

INTRACELLULAR TARGET FOR α-TERTHIENYL PHOTOSENSITIZATION: INVOLVEMENT OF LYSOSOMAL MEMBRANE DAMAGE

Abstract— Intracellular targets for the photosensitizer α-terthienyl (αT) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of αT was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of αT. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, β-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(β-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320–400 nm) irradiation of the cells after uptake of αT and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of αT and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on αT photosensitization. The present results indicated that lysosomes are the primary photosensitization site of αT.     

Influence of photosensitizer solvent on the mechanisms of photoactivated killing of Enterococcus faecalis

This study evaluated the mechanisms involved and the influence of photosensitizer solvent in the killing of Enterococcus faecalis using photodynamic therapy (PDT). Enterococcus faecalis cells incubated with 100 microm methylene blue dissolved in water and in MIX (a mixture of glycerol:ethanol:water) were irradiated with 664 nm diode laser (63.69 J cm(-2)). The effect of PDT on the viability of bacteria, and the functional integrity of cell wall, chromosomal DNA and membrane proteins were analyzed. The bactericidal action of PDT was significantly higher when a MIX-based photosensitizer solvent was used (P<0.001). Fluorimetric and fluorescence microscopy-based analysis showed the functional impairment of E. faecalis cell wall which was significantly higher when a MIX-based photosensitizer solvent was used (P<0.001). PDT with MIX-based photosensitizer solvent showed extensive damage to chromosomal DNA. However, both PDT conditions showed similar trend in the degradation of membrane proteins, although cross-linked proteins were evident only in PDT conducted with MIX-based photosensitizer solvent. The findings from our study showed that PDT destroyed the functional integrity of cell wall, DNA and membrane proteins of E. faecalis. The degrees of damage on these targets were influenced by the photosensitizer solvent used during PDT.     

Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens

The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (10(8) cells per mL) were incubated with hypericin (0-40 μM) for 30 min and followed by light irradiation of 600-800 nm at 5-30 J cm(-2). Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

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.     

PHOTOSENSITIZED INACTIVATION OF E. COLI CELLS IN TOLUIDINE BLUE-LIGHT SYSTEM

E. coli cells were inactivated with visible light in the presence of toluidine blue as a photo-sensitizer. This photodynamic effect was partially protected with α-tocopherol. Not only pH but the concentration of the buffer during irradiation also affected the survival. The addition of osmotic stabilizers such as KCI, glycerol and polyethyleneglycol to the buffer increased the survival. The difference in singlet oxygen production in these reaction mixtures could not be related to these features. Furthermore, the survival was also dependent upon both irradiation temperature and cultivation temperature of the cells. These results with E. coli cells support the notion that one of the primary targets of toluidine blue sensitized photodynamic inactivation is cytoplasmic membrane, although other factors than cytoplasmic membrane also influence the survival of the cells.     

Analysis of the bacterial heat shock response to photodynamic therapy-mediated oxidative stress

Antimicrobial photodynamic therapy (PDT) has recently emerged as an effective modality for the selective destruction of bacteria and other pathogenic microorganisms. We investigated whether PDT induced protective responses such as heat shock proteins (HSPs) in bacteria. Using the photosensitizer Toluidine Blue O (TBO) at sublethal PDT conditions, a seven-fold increase in bacterial HSP GroEL and a three-fold increase in HSP DnaK were observed in Escherichia coli post PDT. Pretreatment with 50°C heat for 30 min reduced PDT killing in both E. coli and in Enterococcus faecalis, with the most pronounced inhibition occurring at 50 μm TBO with 5 J cm(-2) 635 nm light, where E. coli killing was reduced by 2 log(10) and E. faecalis killing was reduced by 4 log(10). Finally, inhibition of the highly conserved chaperone DnaK using a small molecule benzylidene lactam HSP inhibitor potentiated (but not significantly) the effect of PDT at a TBO concentration of 2.5 μm in E. faecalis; however, this effect was not observed in E. coli presumably because inhibitor could not gain access due to Gram-negative permeability barrier. Induction of HSPs may be a mechanism whereby bacteria could become resistant to PDT and warrants the need for further study in the application of dual PDT-HSP-inhibition therapies.     

ACTION OF VISIBLE LIGHT ON ENZYMES IN CELL ENVELOPES OF MICROCOCCUS ROSEUS

Abstract— Envelopes were isolated from the carotenogenic bacterium Micrococcus roseus , which is subject to photodynamic killing in the presence of a photosensitizing dye but not in the absence of such a dye. Envelope preparations contained 88 per cent of the total cellular carotenoids, 20% of the NADH oxidase, 100 per cent of the ATPase and 30% of the succinic dehydrogenase activity. NADH oxidase activity in envelopes was stimulated 2–5-fold by light in the presence of dye; this was followed by inactivation. In the presence of dye, ATPase was inactivated by light and 25 per cent of the succinic dehydrogenase activity was lost. In the absence of dye, responses were extended over a longer period of time, but similar patterns were observed for the three enzymes, indicating that envelopes contain an endogenous photosensitizer(s). Carotenoid-deficient cells were obtained after growth in medium containing diphenylamine. But all three enzymes showed evidence of instability in envelope preparations, indicating that diphenylamine affects membrane structure in addition to inhibiting synthesis of colored carotenoids.     

MECHANISMS OF CITRAL PHOTOTOXICITY

Citral, a monoterpene aldehyde synthesized by several plant genera, has been reported to exhibit antimicrobial activity. For the first time, we report that critral exhibits UV-A (315-400 nm) light enhanced oxygen-dependent toxicity against a series of Escherichia coli strains differing in DNA repair and catalase proficiency. Those E. coli strains carrying a gene leading to catalase deficiency (katF) are particularly sensitized to inactivation by citral and UV-A treatment when compared to catalase proficient strains (katF+). Consistent with these in vivo observations, citral when treated with UV-A in vitro produces H2O2. When tested against Fusarium oxysporum and F. solani, fungal root pathogens of Citrus, enhanced toxicity by citral in the presence of UV-A was demonstrated, while dark toxicity was negligible. When the plasmid pBR322 was treated with citral in the presence of UV-A, a change in conformation from the covalently closed circular to the open circular and, ultimately, the linear form was observed. The change in plasmid conformation corresponded to a reduction in transforming activity. Holding plasmid DNA which had been treated with UV-A light in the presence of citral at 4 degrees C for 22 h in the dark resulted in continued degradation of the DNA and loss of transforming activity. Holding plasmid DNA treated with UV-A or citral alone under identical conditions had no detectable effect on either plasmid conformation or transforming activity.     

THIOPYRONINE- AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY and RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract –We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

Escherichia coli: Dominance of Red Light over Other Visible Light Sources in Establishing Viable but Nonculturable State

In this study, the effect of UV-A and different wavelengths of visible light irradiations combined with or without a photosensitizer (methylene blue, MB) on the establishment of viable but nonculturable (VBNC) state in Escherichia coli was investigated. Survival of the E. coli was investigated by measuring plate counts, respiring cell count (RCC), direct viable count (DVC) and total counts over a period of up to 72 h. The inhibition rates of various light sources in the presence or absence of MB on E. coli in seawater were ranked in the order UV-A>red light>white light>blue light>green light (from greatest to least activation). E. coli survived for 10.2, 19.0, 21.3 and 24.04 h under exposure to red, white, blue and green light and for 6.8 h under exposure to UV-A in the presence of MB according to t ₉₉ . Although the VC declined to undetectable levels in a relatively short time, the RCC showed that some cells were still capable of respiration and, therefore, are assumed to have entered the VBNC phase. This is the first time that red light has been shown to have a stronger effect on E. coli survival and VBNC than white, green and blue light in seawater environment.     

RESEARCH NOTE: THIOPYRONINE-AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY AND RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract: We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

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.     

Polycationic chitosan-conjugated photosensitizer for antibacterial photodynamic therapy

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan-conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7-day-old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.     

MECHANISMS OF CELL KILLING IN PHOTODYNAMIC THERAPY USING A NOVEL in vivo DRUG/in vitro LIGHT CULTURE SYSTEM

Photodynamic therapy of certain neoplasms has emerged as a promising form of cancer treatment. This type of therapy involves the exogenous administration of a photosensitizer with subsequent exposure to light. The ensuing photochemical reaction results in destruction of the tumor. Whether tumor cells are destroyed directly by the photodynamic treatment or indirectly as a result of destruction of the tumor microvascular bed is unknown. To address this question, methods were adapted to test whether combinations of a photosensitizer and light resulted in direct cell killing of precision cut tissue slices placed in culture. The major advantages of this culture system are that photosensitizers are administered in vivo, tissue slices produced in minutes, placed in culture medium, and irradiated in vitro. Any resulting cellular destruction occurs in the absence of a functioning vascular system and indicates that photodynamic therapy acts through a direct cell killing mechanism. Tissue slice viability was monitored by two standard methods: assay for intracellular potassium and morphological examination at the electron microscopic level. The effects of hematoporphyrin derivative and light were examined on tissue slices produced from a prostate adenocarcinoma transplanted into male Copenhagen rats. The data indicate that direct killing of tumor slices occurs and is dependent on the irradiation protocol used.     

PHOTOKILLING OF MICROCOCCUS ROSEUS

Abstract. Previous work showed that the bacterium Micrococcus roseus is killed by photodynamic action when an exogenous photosensitizer is present, but when a photosensitizer is not added the organism survives long exposure to high intensity (22,000ft-c, 348 J/s/m²) white light. Experiments designed to demonstrate the presence of a mechanism to repair damage caused by photodynamic action failed to reveal such a mechanism. However, the organism was killed by light of a very high intensity (32,000ft-c, 506 J/s/m²) in the absence of added photosensitizer, indicating that cells have an effective endogenous photosensitizer(s). Two carotenoid-deficient mutants were killed via photodynamic action more rapidly than the fully pigmented wild-type in the presence or absence of an exogenous photosensitizer. Thus, resistance of M. roseus to photodynamic action is not due to a repair mechanism, nor to lack of an effective endogenous photosensitizer, but to the protective action of carotenoid pigments.     

CONFORMATIONAL CHANGES OF BOVINE LENS CRYSTALLINS IN A PHOTODYNAMIC SYSTEM

Abstract— Conformational changes of bovine lens crystallins in a photodynamic system generating singlet oxygen, have been investigated. The formation of intersubunit crosslinks was observed in all three classes (α-, β and γ-) of crystallins by irradiation in the presence of the photosensitizer methylene blue. Near-UV circular dichroism (CD) spectra of the crystallins were significantly altered by irradiation under these conditions, indicating changes in tertiary structure but the far-UV CD remained unchanged suggesting that the secondary structure ((β-sheet conformation) remains unchanged. Significant changes in the absorption and fluorescence spectra were also observed. Measurement of total sulfhydryl content showed a decrease of 27%, 50% and 37% for α-, β- and γ-crystallins respectively, after irradiation. Fluorescence lifetime measurements of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine-labeled crystallins showed a significant decrease of the lifetime of the major decay components of the label bound to sulfhydryl groups of α- and γ-crystallins, but showed no change in the microenvironment of the sulfhydryl groups of β-crystallin. The results are consistent with the microenvironments of the tryptophan and sulfhydryl groups predicted from sequence studies.     

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.