PHOTOSENSITIZED INACTIVATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine was found to sensitize cultured Chinese hamster cells upon exposure to white fluorescent light. Elimination of wavelengths below 370 nm did not reduce the effect significantly, indicating that the effective wavelengths were those absorbed by the Q band (600–700 nm) of phthalocyanine. The magnitude of the photosensitizing effect increased with the dye concentration and the time of its contact with the cells prior to light exposure. Although photosensitization was drastically reduced in the absence of oxygen, the lack of effect of glycerol and D₂0 during exposure suggests that neither hydroxyl radicals nor ¹O₂ are responsible for the cytotoxic response. The efficiency of the photosensitized induced cell killing did not vary with the position of the cells in the cell cycle, in contrast to exposure to X-rays. The improved spectral properties, the reported low toxicity and the selective retention by neoplasms, make phthalocyanines promising candidates for use in photodynamic therapy of cancer.     

THE USE OF SPIN LABEL OXIMETRY IN THE STUDY OF PHOTODYNAMIC INACTIVATION OF CHINESE HAMSTER OVARY CELLS

Spin label oximetry has been used to study the effects of photosensitizer hematoporphyrin on oxygen consumption in Chinese hamster ovary cells. These measurements demonstrate that, in the presence of the sensitizer, there occurs (i) an increase in total oxygen consumption during irradiation and (ii) a decrease in oxygen consumption following irradiation. These results are attributed to the effects of photosensitized oxygen consumption and decreased cellular respiration due to cell inactivation. Thus the inhibition of oxygen consumption after irradiation was shown to correlate with decreased cell survival measured in cell culture experiments. Possible mechanisms of inactivation and extensions of the oximetric approach are discussed.     

PTHALOCYANINE-INDUCED PHOTODYNAMIC CHANGES OF CYTOPLASMIC FREE CALCIUM IN CHINESE HAMSTER CELLS

Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca2+], from about 0.2 microM to 1 microM within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca2+]i. These findings raise the possibility that changes in [Ca2+]i after photodynamic treatment may be relevant to cell killing and/or other biological responses of the cells, e.g. release of eicosanoids.     

THE PHOTODYNAMIC EFFECTS ONV–79 CHINESE HAMSTER CELLS

Abstract— Holding of acriflavine sensitizedV–79 cells in growth medium before visible light exposure decreases inactivation by visible light. The decrease depended upon the period of holding, indicating that there was release of cellular dye during this period. Exposures to visible light were done in two conditions: (a) with no dye in the medium during visible light exposure (washed) and (b) with dye in the medium during exposure (unwashed). Caffeine was found to slightly increase the sensitivity of the cells to visible light in the washed condition, whereas, in the unwashed condition no such effect was observed. Interaction studies with far UV did not reveal any correlation between photodynamic damage and UV damage. Visible light exposure of acriflavine sensitized cells was found to be mutagenic, as studied from the induction of 8-azaguanine resistant mutants. Inhibition of singlet oxygen production by sodium azide suppressed the induction of mutants. All these, taken together, have been discussed with respect to the relative importance of DNA and non-DNA damage in the photodynamic action of acriflavine.     

PROTECTION BY THE FLUORIDE ION AGAINST PHTHALOCYANINE-INDUCED PHOTODYNAMIC KILLING OF CHINESE HAMSTER CELLS

When a dilute F- solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCl, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F-. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA-protein crosslinks caused by light and this sensitizer were also inhibited by F-. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA-protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3-N(CH3)2 were not inhibited by F-. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F-, and that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA-protein crosslink formation indicates that the effects of F- can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F-.     

PHOTODYNAMIC INACTIVATION OF YEAST CELLS SENSITIZED BY HEMATOPORPHYRIN

Abstract— Yeast cells are inactivated by treatment with hematoporphyrin and light. The inactivation is mainly mediated by singlet oxygen. The quantum yield of singlet oxygen increases with increasing pH, while the efficiency of cellular inactivation decreases with increasing pH. Cells in the stationary phase are much more resistant to the treatment than cells in exponential growth. Membrane damage seems to be the main determining step in the photoinactivation.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m² of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.     

EFFECT OF PHOSPHATE AND CHLORIDE ON THE PHOTODYNAMIC INACTIVATION OF YEAST CELLS

Abstract— Yeast cells are inactivated by treatment with hematoporphyrin and light. The inactivation, which is mediated by singlet oxygen (¹O₂), is enhanced by the presence of phosphate and chloride. Neither phosphate nor chloride has any influence on the yield of ¹O₂. Possible mechanisms for the enhancement are briefly discussed.     

STIMULATORY AND INHIBITORY EFFECTS OF IRON ON PHOTODYNAMIC INACTIVATION OF LEUKEMIA CELLS

The influence of exogenous iron on merocyanine 540 (MC540))sensitized photoinactivation of leukemia cells has been investigated. Irradiation of murine L1210 or human HL-60 cells (-lOh/mL in 1 % serumiRPM1 medium) with broadband visible light in the presence of MC540 (2 pIM) resulted in a progressive loss of clonally assessed cell viability. When added to cells 30 min before irradiation, the low polarity chelate, ferric 8-hydroxyquinoline [Fe(HQ), 0.5 *MI stimulated dye-sensitized pho- tokilling, whereas high polarity chelates such as ferric 8-hydroxyquinoline-5-sulfonate [Fe(HQS)2, 0.5 p M] or ferric ethylenediaminetetraacetate (Fe.EDTA, 0.5 FM) had no effect. A striking reversal of Fe(HQ),-enhanced photokilling was observed upon increasing the preirradiation incubation time with Fe(HQ)2 such that a marked resistance (relative to non-iron-treated controls) was evident after 24 h. Cells exposed for 24 h to Fe(HQS), or Fe.EDTA showed similar or even greater resistance to photo-killing. Like phototoxicity, H,O,-induced cytotoxicity was enhanced after a 30 min exposure of cells to Fe(HQ)2 but strongly repressed after 24 h. Immunoblot (western) analysis, using a polyclonal antibody to ferritin, revealed that cells exposed to Fe(HQ): for 24 h contained at least 12 times as much ferritin heavy chain as non-Fe(HQ)?-treated controls. Preincubating cells with emetine, an inhibitor of protein synthesis, prevented both ferritin induction and the development of hyperresistance. These findings, along with the observation that exogenous apoferritin protected LI 2 10 cells against photokilling, suggest a possible role for ferritin in iron-stimulated photoresistance. It is conceivable that in photodynamic treatment of tumors, certain cells might resist inactivation via this mechanism, a possibility that has not been recognized heretofore.     

GENETIC TOXICOLOGY OF THE PHOTOSENSITIZATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.     

RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 and CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.     

PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN

Abstract— It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo-dynamic inactivation of hen egg white lysozyme by eosin, using D₂O to enhance the solvent-induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air-saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H₂O to D₂O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re-examination of earlier work on eosin-sensitized photo-oxidation of I", it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

EFFECTIVE PHOTODYNAMIC ACTION BY RHODAMINE 123 LEADING TO PHOTOSENSITIZED KILLING OF CHINESE HAMSTER OVARY CELLS IN TISSUE CULTURE AND A PROPOSED MECHANISM

The effectiveness of rhodamine 123 (R123) as a photosensitizer of cell killing is relatively low and correlates with its inefficient production of singlet oxygen. The known selective retention of R123 in the mitochondria of epithelially derived carcinoma cells, however, is a selective feature that could lead to a more useful therapeutic ratio if photosensitizing effectiveness could be increased. Chinese hamster ovary (CHO) cells in tissue culture were therefore exposed to R123 shortly before and during illumination under conditions controlled for oxygen concentration and temperature. Effective photosensitization of cell killing, as judged by colony formation, was produced by 95% but not by 19% O₂ during illumination of cells at 5d?C or 37d?C, and this was additionally enhanced at the sublethal temperature of 42d?C. Two CHO cell lines were examined; one line, CHO-AA8, was proficient in the repair of DNA damage and the parent to the second line, CHO-EM9, that was deficient in the repair of DNA strand breaks. Cells of both lines incorporated R123 to a similar degree and were similarly photosensitized by the presence of igh oxygen concentration. Furthermore, plasma membrane damage as judged by teh exclusion of trypan blue was not observed immediately after illumination in the presence of R123, but was seen in the presence of meso-tetra-(4-sulfonatophenyl)-porphine (TPPS₄). The extent of damage to the plasma membrane by TPPS₄ was greater in the presence of 95% compared to 19% O₂ during illumination. Photodynamic action at the level of teh plasma membrane appears to contribute to photosensitization by TPPS₄ but not by R123 soon after exposure of cells to these sensitizers. It is hypothesized that photodynamic action by R123 is the primary mechanism causing the observed photosensitization of cell killing, and that mitochondria are teh site of photosensitized damage responsible for this killing.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

LASER-LIGHT INDUCED CHROMOSOME ABERRATIONS IN CHINESE HAMSTER CELLS

Wild-type Chinese hamster cells CHO Kl and their radiosensitive mutant xrs5 were irradiated at 308 nm, using light pulses of a XeCl excimer laser with total energy fluences of 0.1 kj/m² to 4.08 kj/m². Chromosome-type and chromatid-type chromosome aberrations have been observed at pulse irradiances of 2.5 × 10⁷ W/m² and 1.7 × 10⁸ W/m², indicating that in mammalian cells DNA double-strand breaks occur already in this irradiance range. The results obtained with laser irradiation are compared with X-ray irradiation.     

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET-IRRADIATED CHINESE HAMSTER CELLS

Abstract —As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer-containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer-containing DNA is replicated in ultraviolet-irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.