Protection against light-activated photofrin II killing of tumor cells by nitroimidazoles.

Nitroimidazoles are good quenchers of triplet state porphyrins in chemical systems, thereby inhibiting singlet oxygen formation and type II photodynamic reactions. Photobiological studies were performed with EMT-6 tumor cells in vitro utilizing Photofrin II (PII) in combination with etanidazole (ETAN), misonidazole (MISO), and trifluoromisonidazole (TF-MISO). After short-term (1 h) exposure of cells to PII, 5 mM ETAN and MISO had no effect on photoinactivation while 5 mM TF-MISO had a small but significant protective effect. When the intracellular oxygen level was equilibrated with 0.3% oxygen in the gas phase, all three nitroimidazoles produced significant photoprotection at concentrations as low as 0.3 microM. After long-term (24 h) exposure of cells to PII, all three nitroimidazoles demonstrated large photoprotective effects under both aerobic and 0.3% oxygen conditions. At equal concentrations of nitroimidazole, photoprotection was greatest for the most lipophilic compound (TF-MISO) and least effective for the most hydrophilic compound (ETAN). These studies suggest that nitroimidazoles can quench triplet state porphyrins (within cells) to reduce intracellular concentrations of singlet oxygen, the putative toxin in PII photoinactivation. In addition, after long-term exposures to PII when porphyrins have partitioned into cellular membranes and lipid environments, the lipophilicity of this class of photoprotector correlates with effectiveness in these mammalian cells.     

Photodynamic effects of Photofrin II on cell division in human NHIK 3025 cells

Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.     

Spin-trapping of free radicals during phthalocyanine photosensitization of lymphoma cells in vitro.

Spin-trapping and electron spin resonance (ESR) spectroscopy were used to detect free radicals generated during light exposure of lymphoma cells sensitized in vitro by metallotetrasulfophthalocyanines (Al-PcS4 and Zn-PcS4). 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and alpha-phenyl-beta-tert-butylnitrone (PBN) were used as spin-trapping agents. Hydroxyl radical spin-adducts were detected under conditions of both extracellular and intracellular photosensitization. In addition, organic radicals of different origin and/or variable yields were trapped, depending on the photosensitization conditions and the spin-trap used. For comparison, analogous experiments were carried out with another tumor-localizing photosensitizer, Photofrin II.     

Proton magic angle spinning NMR reveals new features in photodynamically treated bacteria

The bacterium Propionibacterium acnes is light-sensitive due to porphyrin-induced photosensitization. The light sensitivity increases with incubation of 5-aminolevulinic acid, ALA. For the first time, 1H magic angle spinning NMR spectroscopy is used to describe the photoinduced changes in the bacterium after ALA incubation. Successful photosensitization was performed with light-emitting diodes in the blue and red regions (430 and 654 nm, respectively). The irradiation setup, suitable for irradiation of bacterium suspensions in petri dishes is described. For NMR studies blue light diodes with about 90 micromol/m2s were chosen. After blue light irradiation, the endogenous glycine betaine, proline, glutamate and choline levels in P. acnes decreased with increasing irradiation time. For sublethal light doses (50% survival fraction), the endogenous glycine betaine level decreased 80% on average. The corresponding percentages for proline, choline and glutamate were about 40, 25 and 10, respectively. It is hypothesized that the irradiation, inducing porphyrin photosensitization amplified by ALA incubation, leads to elimination of the osmolyte glycine betaine and possibly also proline by so-called regulatory volume decrease (RVD) mechanisms. These mechanisms are known to be active in several prokaryotic and eukaryotic cells when exposed to hypotonic stress. They are also known to be present in several eukaryotic cells during photodynamic therapy (PDT) exposure leading to hypotonoc stress. The findings contribute to the knowledge of the inactivation mechanisms of P. acnes in photosensitization, and could therefore be of interest in the efforts to use PDT as treatment of the acne disease.     

Recent developments in and intercomparisons of acute and chronic bioassays and bioindicators

The ultimate goal of toxicity testing is to monitor or predict the effects of single compounds, elements or mixtures on the long-term health of individual organisms, populations, communities and ecosystems. Unfortunately, one does not always have all of the information required to determine the long-term or chronic effects of toxicants on the survival, growth or reproduction of aquatic organisms. For this reason, the chronic effects of toxicants are often inferred or estimated from observations made during short-term or or acute field or laboratory studies, which may be conducted at greater concentrations of toxicant. The observations made in the short-term studies are then related to the chronic effects by some statistical relationship. There are basically two approaches: 1) The long-term effects on a parameter, such as survival (lethality.) are predicted from observations on the same parameter, during short-term exposures; 2) Alternatively, the response of one parameter to long-term exposures of a toxicant can be predicted from the short-term responses of a different parameter. In this report we present several different examples of both types of methods for estimating chronic responses from information on more short-term responses and discuss the rationale, advantages and disadvantages of each. We also report on two biochemical indicators; energetic substrates and RNA/DNA ratio. These indicators both act as sensitive, integrative measures of sublethal effects of contaminants during both acute and chronic exposures.     

Haematoporphyrin derivative photosensitization and gamma-radiation damage interaction in Chinese hamster ovary fibroblasts

The interaction of haematoporphyrin derivative (HPD) photosensitization and gamma-irradiation was studied with regard to clonogenicity of Chinese hamster ovary (CHO) fibroblasts. Exposure to either treatment alone resulted in shouldered response curves. Exposure to 4.2 Gy gamma-radiation immediately before graded doses of visible light had no effect on the shape of the visible-light survival curve; similarly, exposure to 8.75 kJ/m2 light immediately before graded doses of gamma-radiation had no effect on the shape of the gamma-radiation response curve. These data indicate that damage due to gamma-radiation and HPD photosensitization did not interact, suggesting that the mechanisms of cell killing are different.     

Electric Field-Assisted Delivery of Photofrin to Human Breast Carcinoma Cells

The influence of electroporation on the Photofrin uptake and distribution was evaluated in the breast adenocarcinoma cells (MCF-7) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent channels in vitro. Photofrin was used at a concentration of 5 and 25 μM. The uptake of Photofrin was assessed using flow cytometry and fluorescence microscopy methods. Cells viability was evaluated with crystal violet assay. Our results indicated that electropermeabilization of cells, in the presence of Photofrin, increased the uptake of the photosensitizer. Even at the lowest electric field intensity (700 V/cm) Photofrin transport was enhanced. Flow cytometry results for MCF-7 cells revealed ~1.7 times stronger fluorescence emission intensity for cells exposed to Photofrin and electric field of 700 V/cm than cells treated with Photofrin alone. Photofrin was effective only when irradiated with blue light. Our studies on combination of photodynamic reaction with electroporation suggested improved effectiveness of the treatment and showed intracellular distribution of Photofrin. This approach may be attractive for cancer treatment as enhanced cellular uptake of Photofrin in MCF-7 cells can help to reduce effective dose of the photosensitizer and exposure time in this type of cancer, diminishing side effects of the therapy.     

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.     

Photosensitivity of DNA replication and respiration to haematoporphyrin derivative (photofrin II) in mammalian CV-1 cells

DNA synthesis, as well as respiration, has been studied in CV-1 cells incubated with 5 or 25 micrograms/cm3 haematoporphyrin derivative Photofrin II (PF II) for 1, 24 or 48 h and then irradiated with various doses of UVA light (365 nm). The impairments of DNA synthesis increased with the duration of incubation with the porphyrin, its concentration and the dose of irradiation. The cellular consumption of oxygen is also inhibited by the treatment, but less severely. In the case of the higher PF II concentration (25 micrograms/cm3), the impairment of DNA synthesis after illumination seems to be mainly due to 3HTdR transport inhibition. This effect can be related to plasma membrane damage as shown by lactate dehydrogenase leakage. At 5 micrograms/cm3 PF II, DNA synthesis inhibition is observed even after short exposure to PF II and light without 3HTdR transport impairment. In that case, DNA and/or mitochondrial photodamage may explain the inhibition.     

Studies on the photochemical and photocytotoxic properties of the new PDT photosensitizer aluminum sulfonated phthalocyanine.

The properties of photosensitization of sulfonated aluminum phthalocyanine (ALSPC), a new photosensitizer of potential use in cancer photodynamic therapy (PDT) was studied on both the molecular and cellular levels. The mechanism of ALSPC photosensitization on the molecular level was investigated by testing its efficiency of singlet oxygen (1O2) production, using the method of tryptophan degradation and that of ESR spectroscopy and observing the enhancing effect of D2O and the quenching effect of NaN3. Results of all these experiments confirmed the important role of the Type II or 1O2 mechanism in ALSPC photosensitization. In our in-vitro experiments, ALSPC's incorporation into cells and its photocytotoxic effect were investigated on a human liver cancer cell line. The cell incorporation was illustrated by the laser-excited fluorescence spectra emitted both from cell homogenate and cell monolayers incubated with ALSPC aqueous solution. The position of fluorescence peak observed, implied that ALSPC exists in the cells mainly as monomers. The efficiency of cell killing of ALSPC photosensitization was estimated by counting surviving cells with the method of trypan blue staining and by the method of radioisotope labelling. Experiments using the latter method also showed DNA damage caused by ALSPC photosensitization.     

Photosensitization of lymphoblastoid cells with phthalocyanines at different saturating incubation times

Photodynamic therapy of cancer is a promising treatment based on the tumor-specific accumulation of photosensitizers followed by irradiation with visible light which induces tumor cell death. The effect of different preincubation times on the photosensitization efficiency of the phthalocyanines AlPc and AlPcS₄ was investigated in lymphoblastoid CCRF-CEM cells under conditions that allow maximal uptake of the sensitizers. First, the time course for the uptake of AlPcS₄ and AlPc by CCRF-CEM cells and by the pheochromocytoma PC12 cells was compared. The uptake of AlPcS₄ by CCRF-CEM cells was not significantly different after 6 h or 24 h incubation, but the photosensitization efficiency of the phthalocyanine was much higher when a 24 h preincubation period was used, with a fluence rate of 5 mW/cm². However, for a fluence rate of 10 mW/cm², the photosensitization efficiency of AlPcS₄ was almost completely independent of the preincubation time (6 h vs. 24 h) with the phthalocyanine. When the cells were preincubated with 1 mol/L AlPc for 10 min or 6 h, which allows the same accumulation of sensitizer by the cells, no significant effect of the incubation time on the photodynamic inactivation of CCRF-CEM cells was observed, with fluence rates of 5 mW/cm² or 10 mW/cm², for different light doses. Confocal fluorescence microscopy studies did not reveal differences in the localization of the phthalocyanines after maximal uptake was reached. The results show that the preincubation time with AlPcS₄, after the maximal uptake is reached, affects cell growth to an extent depending on the fluence rate used, and this effect was not due to a major redistribution of the sensitizer during incubation. However, this was not observed when AlPc was used.     

Cell photosensitization by 5-iminodaunomycin activated with red light

5-Iminodaunomycin, an anthracycline antitumor drug exhibiting an absorption peak at 595 nm, is shown to photosensitize in vitro cell kill. The photoactivation is performed irradiating the culture dishes during the incubation with the drug for 2 h with 34 mW/cm2 intensity, that is with light doses of up to 245 J/cm2. Long-term effects of administering 50 ng/ml and light for 2 h are studied in terms of growth curves. We show that photoactivation enhances the dark toxicity by a factor of about 10. Immediate cell death is produced by irradiating the cells in the presence of higher drug concentrations (e.g., 1000 ng/ml) which, however, are not toxic in the short term if administered in the dark. The viable cell percentage decreases at increasing light doses, being about 0.6% at the maximum dosage. Administering lower light doses, such as 30 J/cm2, which corresponds to an exposure duration of 15 min, has a short-term effect on the cell survival that strongly depends on the timing of the exposures within the incubation period.     

Modelling the photosensitization-based inactivation of Bacillus cereus

Aims:  To study and to develop a model for the photo-destruction of the foodborne pathogen Bacillus cereus , initially treated with a precursor of endogenous photosensitizers (5-aminolevulinic acid, ALA).
Materials and methods:  The cells were incubated in the presence of ALA (3 or 7·5 mmol l⁻¹) for incubation times ranging from 2 to 60 min, inoculated onto the surface of LB Agar plates and submitted to light irradiation. The Weibull model was used to describe the survival curves of B. cereus . Quadratic equations were used to describe the effects of ALA concentration and incubation time on the Weibull model parameters.
Results:  ALA-based photosensitization proved to be an effective tool for inactivation of B. cereus . The decrease in viable counts observed after 20 min of irradiation, ranged from 4 to 6 log CFU g⁻¹.
Conclusions:  The developed model proved to be a parsimonious and robust solution to describe the observed data.
Significance and Impact of the Study:  The study demonstrates the effectiveness of photosensitization on B. cereus on agar plates. The model developed may be useful to optimize inactivation treatments by photosensitization.     

Modification by cis-diamminedichloroplatinum (II) of the radiation dose-response curve for intestinal crypt cells in mice

The effect of cis-diamminedichloroplatinum (II) (c-DDP) on the shape of the radiation dose-response curve for mouse duodenal crypt cells was investigated. A priming X-ray dose was followed 18 h later by graded test doses (single doses or five equal fractions at 3-h intervals) with or without c-DDP. Curves were fitted by a linear quadratic (LQ) relationship. The drug modified the dose-response curve by enhancing both the alpha and the beta terms. Repair kinetics were analyzed in split-dose experiments. c-DDP caused a minor, nonsignificant decrease in the rate of repair after irradiation. The survival ratio after split-dose irradiation, when the same X-ray doses were given, was actually slightly increased by the drug. This paradoxical effect can be explained by the fact that c-DDP mainly increased the beta term in the LQ relationship. There was no significant increase in crypt cell survival when split-drug doses were given alone at increasing intervals, suggesting no cellular repair after c-DDP treatment. The data are discussed in the light of the recently proposed "lethal and potentially lethal" (LPL) unified repair model of Curtis.     

No correlation between DNA strand breaks and HPRT mutation induced by photochemical treatment in V79 cells

DNA strand breaks, measured by alkaline elution, and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutation were studied in V79 cells after photochemical treatment (PCT) or exposure to X-rays. Cells were incubated with the photosensitizers Photofrin II (PII) and three closely related porphyrins tetra-(3-hydroxyphenyl) porphyrin (3THPP), meso-tetra-(4-sulfonatophenyl) porphine (TPPS₄) and meso-tetra-(N-methyl-4-pyridyl) porphine (TMPyPH₂). These dyes are assumed to act on cellular targets mainly via singlet oxygen when excited by light. While the hydrophilic TPPS₄ and TMPyPH₂ did not photoinduce mutants to any significant extent, both lipophilic dyes, 3THPP and PII, were significantly mutagenic when excited by light. On the other hand, TPPS₄ was the most efficient sensitizer of alkali-labile DNA strand breaks, while TMPyPH₂ did not induce any significant amount of either type of DNA damage. Surprisingly, no correlation between the two parameters was found for PCT, either after exposures inactivating 50% of the cells or after exposures inactivating 90% of them. The lack of correlation between the yields of DNA strand breaks and of mutants could not be explained by differences in the intracellular localization pattern of the dyes.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations. II. Photoreactivation and killing of rat kangaroo cells (Potorous tridactylus) and Herpes simplex virus-1 by exposure to fluorescent "white" light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridacylus) has been studied at wavelengths greater than 375 nm from either fluorescent "white" light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths greater than 475 nm, and even greater than 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-inactivated Herpes simplex Virus-1 (HSV-1) after infection leads to PR effects resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The "fluence-reduction factor" of PR, which is greater than 3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight greater than 375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.     

Photodynamic effect of deuteroporphyrin IX and hematoporphyrin derivatives on single neuron

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.     

Effect of dibromothymoquinone on Chlorophyll <Emphasis Type="Italic">a</Emphasis> Fluorescence in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells incubated in complete or sulfur-depleted medium

The effect of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. Dibromothymoquinone was shown to affect differently control cells incubated in complete medium and S-starved cells. The fluorescence yield in the control suspension considerably increased in the presence of the inhibitor. Presumably, this can be due to inactivation of protein kinase, as a result of which part of light-harvesting complex II that could have diffused from the stacking zone of the membrane into the lamellar zone towards photosystem I remains close to photosystem II. In S-starved cells, whose photosynthetic apparatus is in state 2, the fluorescence level declines in the presence of dibromothymoquinone. The JIP testing of induction curves (O-J-I-P fluorescence transient) suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to the control, while in the starved cells it acts predominantly as an electron acceptor.     

A practical test for in vitro evaluation of photosensitization: Assessment with hematoporphyrin derivative (HPD)

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities.The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.Abbreviations DHE Dihematoporphyrin Ether - FCS Fetal Calf Serum - HPD Hematoporphyrin Derivative - PDT Photodynamic Therapy     

The interaction of visible and UV-B light during photodamage and repair of Photosystem II

In order to understand the mechanism of photodamage induced by solar radiation under natural conditions, we studied the interaction of visible and ultraviolet-B light in the inactivation and repair of the Photosystem II complex by using oxygen evolution and flash-induced chlorophyll fluorescence measurements. In isolated spinach thylakoids and Synechocystis 6803 cells, in which de novo protein synthesis is blocked by lincomycin, photodamage of Photosystem II by visible and UV-B light is characterized by linear semilogarithmic inactivation curves for both separate and combined illumination protocols. The extent of PS II inactivation obtained after combined illumination can be well simulated by assuming independent damaging events induced by visible and UV-B photons. In intact Synechocystis cells capable of protein repair, simultaneous illumination by visible and UV-B light impairs Photosystem II activity to a smaller extent than expected from the independent damaging events. This protective effect is pronounced at low visible light (130 μE m⁻² s⁻¹), but becomes negligible at high intensities (1300 μE m⁻² s⁻¹). Exposure of intact Synechocystis 6803 cells to direct sunlight leads to a rapid inactivation of PS II, accompanied by the accumulation of donor side inhibited centers. This phenomenon, which shows the impairment of the manganese cluster of water oxidation was not observed when the ultraviolet components of sunlight were filtered out. We conclude that visible and UV-B photons inactivate PS II via non-interacting mechanisms, which affect different target sites. In intact cells, the two spectral regions do interact, and results in synergistically enhanced protein repair capacity when UV-B radiation is accompanied by low intensity visible light, which provides protection against photodamage. However, this ameliorating effect becomes insignificant at high light intensities characteristic of direct sunlight. This revised version was published online in June 2006 with corrections to the Cover Date.