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Photosensitization of Escherichia coli and Pseudomonas aeruginosa cells by deuteroporphyrin (DP) is shown to be possible in the presence of the polycationic agent polymyxin nonapeptide (PMNP). Previous studies established complete resistance of Gram-negative bacteria to the photodynamic effects of porphyrins. The present results show that combined treatment of E. coli or P. aeruginosa cultures with DP and PMNP inhibit cell growth and viability. No antibacterial activity of PMNP alone could be demonstrated and cell viability remained unchanged. Spectroscopically, PMNP was found to bind DP, a mechanism which probably assists its penetration into the cell's membranes. Insertion of DP into the cells was monitored by the characteristic fluorescence band of bound DP at 622 nm. Binding times were 5-40 min and the extent of binding increased with decreasing the pH from 8.5 to 6.5. DP binding constants, as well as the concentrations of PMNP which were required for maximal effect on the various Gram-negative bacteria, were determined fluorometrically. By the treatment of DP, PMNP and light the growth of E. coli and P. aeruginosa cultures was stopped and the viability of the culture was dramatically reduced. Within 60 min of treatment the survival fraction of E. coli culture was 9 x 10(-6) and that of P. aeruginosa was 5.2 x 10(-4). Electron microscopy depicted ultrastructural alterations in the Gram-negative cells treated by DP and PMNP. The completion of cell division was inhibited and the chromosomal domain was altered markedly.

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In the present study we examined the production of high amounts of porphyrins upon induction by delta-aminolevulinic acid (ALA) in 9 bacterial strains. This was performed by solely inducing the porphyrin biosynthesis pathway. Four of the strains were Gram positive bacteria and five were Gram negative strains. All strains, except Streptococcus faecalis, produced porphyrins when incubated in PBS with 0.38 mM ALA for 4 h. Excess porphyrin production was excreted to the medium. Gram positive bacteria exhibited fluorescent emission peaks at 622 nm for the endogenous and 617 nm for the excreted porphyrins. Gram negative bacteria exhibited a 630 nm emission peak for the endogenous and a 615 nm emission peak for the excreted extracellular porphyrins. Upon illumination of the ALA induced Staphylococcal strains with 407-420 nm blue light, a decrease of five orders of magnitude was demonstrated with a light dose of 50 J cm(-2). Total eradication of the Staphylococcal strains could be achieved with a 100 J cm(-2) dose, which resulted in a decrease in viability of seven orders of magnitude. The viability of all the induced Gram negative strains and B. cereus decreased by one or two orders of magnitude upon illumination with 50 and 100 J cm(-2), respectively. This difference in the photoinactivation rate was found to be due to the distribution and amounts of the various porphyrins in the bacterial strains. The predominant porphyrin in the Staphylococcal strains was coproporphyrin (68.3-74.6%). In the Gram negative strains there was no predominant porphyrin and the porphyrins found were mostly 5-carboxyporphyrin, uroporphyrin, 7- carboxyporphyrin, coproporphyrin and protoporphyrin. In the B. cereus(Gram positive) strain the predominant porphyrin was uroporphyrin (75.8%). Although the total production of porphyrins in the Gram negative bacteria was higher than in the Staphylococcal strains, the amount of coproporphyrin produced by the latter was twice to three times higher than in the Gram negative strains. The extracellular excreted porphyrins did not contribute to the photoinactivation in any of the tested strains. Significant decreases in the Na(+) and K(+) content were detected in induced S. aureus after illumination while only small changes were observed in E. coli B. The green fluorescent protein within the cytoplasm of induced E. coli strains was only partially disrupted (by 60% only). These results indicate a partial yield of the effects generated by (1)O(2) radicals resulting from the photoinactivation of Gram negative bacteria and a successful generation of the same effects in the Staphylococcal strains.

Cancer therapy ideally should be based on high selectivity of the therapeutic agent for the transformed cells, low specificity for normal tissues and high cytotoxic efficiency to the target tumour. These requirements are partially fulfilled by haematoporphyrin derivative (HPD) phototherapy.The method is based on the capability of porphyrins to be selectively localized in malignant tumours. Light activation of the localized porphyrin induces damage to mitochondria, cellular organelles, membranes, DNA, and specific proteins by singlet oxygen, produced under aerobic conditions and possibly by hydroxyl radicals (Reviewed by Moan, 1986;& Van Steveninck et al., 1986). Photodynamic therapy is based on administration of an HPD solution to the cancer patient and later on, illumination of the tumour by a 630nm laser light beam. The treated area undergoes necrosis, with minimal changes in the surrounding tissues (Dougherty, 1984; Land, 1984;Berenbaum et al., 1982).Although HPD is quite an effective tumour localizer, the trend is to synthesize new porphyrin-compounds with higher tumour localizing capacities, and being chemically well defined, to overcome the main problem of HPD viz. its complex porphyrin composition and aggregation state (Kessel & Chou, 1985;Evensen et al., 1984). Recently, a variety of new molecules with good tumour localization and sensitization properties were introduced into experimental systems, like haematoporphyrin di-ethers (Rimington et al., 1987) and chlorin-porphyrin ester . Uroporphyrin I was reevaluated mainly for diagnostic applications (El-Far & Pimstone, 1986). On the other hand, it is well known that the natural protoporphyrin is an excellent photosensitizer, inducing haemolysis and light sensitivity of the skin in porphyric patients (Meyer & Schmid, 1978). It is a poor tumour localizer despite its high photo-activity potential on in vitro incubated cells (Malik & Djaldetti, 1980). In addition, protoporphyrin is biosynthesized in low amounts, by all tumour cells, as well as non-transformed tissues, while in specific transformed cells such as erythroleukaemia, it can be produced more efficiently (Marks & Rifkind, 1978). In their proerythroblastic phase the enzymatic activity of porphyrin biosynthesis was found to be constitutive, except for the first enzyme, the ALA-synthase which is the rate limiting step of the pathway and is inducible (Sassa, 1976), and iron uptake from transferrin (Lasky et al., 1986). Therefore, in order to induce porphyrin synthesis by erythroblasts, exogenous 5-ALA must be supplied to circumvent the first limiting enzyme .Mice injected with 5-ALA showed porphyrin production in the skin, an effect similar to that of 5-ALA in cultured Friend erythroleukaemic cells (Pottier et al., 1986). From both these systems it can be concluded that the cellular concentration of porphyrin can be increased by exogenous addition of the precursor for porphyrins.The purpose of the present study was to determine whether endogenous porphyrins produced from 5-ALA erythroleukaemic cells ca...

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.

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Exposure of mouse thymocytes to dopamine caused apoptosis (programmed cell death). This was manifested by cellular condensation and membrane damage shown by flow cytometry measurements and scanning electron microscopic study. Dopamine also affected thymocytic nuclei and their genomic DNA integrity. Most of the DNA molecules accumulated in a subdiploid peak in flow cytometry analysis, indicating DNA fragmentation to small particles. DNA analysis showed the typical pattern of 'DNA ladder' caused by internucleosomal DNA cleavage. X-ray microanalysis of the cellular elements of dopamine-treated cells showed elevation of sodium (Na), chloride (Cl) and calcium (Ca) peaks, accompanied by reduction in phosphate (P) concentrations. Comparison of the potassium (K) and P concentrations showed significant differences between the two major death processes: necrosis (induced by exposure to sodium azide (NaN3)) and apoptosis (induced by dopamine). High concentrations of K indicated cell viability while reductions in P and elevations in Ca levels were found to be typical of apoptotic cell death. The antioxidant dithiothreitol (DTT) suppressed dopamine-induced apoptosis in thymocytes, suggesting that its toxicity may be mediated via generation of reactive oxygen radicals. Our study suggests that under certain circumstances, dopamine and/or its metabolites, may induce a process of apoptotic cell death of the dopamine-producing cells in the substantia nigra. Increased accessibility of dopamine to the nigral cell nucleus or inability to scavenge excess free radicals generated from dopamine oxidation triggering programmed cell death, may cause the progressive nigral degeneration in Parkinson's disease.