INT . J . RADIAT. BIOL .,

1992,

VOL .

61,

NO .

6, 767-772

The effect of fluoride on photodynamic-induced fluorescence changes of aluminium phthalocyanine in Chinese hamster cells

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E. BEN-HUR*, J . F . NAGELKERKEt, T . M . A. R . DUBBELMAN$ and J. VAN STEVENINCK§ (Received 11 September 1991 ; revision received 9 December 1991 ; accepted 18 December 1991)

Abstract . Fluence-dependent changes in the fluorescence of aluminium phthalocyanine (A1Pc) were measured in Chinese hamster ovary (CHO) cells using digital fluorescence microscopy of single cells and spectrofluorimetry of cell suspensions . During illumination the fluorescence initially increased and later progressively decreased . In the presence of fluoride, which protects against phototoxicity of AIPc by forming a fluoroaluminium complex, there was no initial increase in fluorescence : it decreased about 10 times faster than in the absence of fluoride . Qualitatively similar results were observed using single-cell fluorescence microscopy, which also showed the dye to be mostly localized in cytoplasmic organelles and membranes . The pattern of localization did not change during illumination . Concomitant assays of dye extracted from cells revealed little photodegradation that could not account for the fluorescence changes. The absorption spectra of AlPc-loaded cells showed some aggregation of the dye prior to light exposure . During illumination the dye was initially monomerized and subsequently progressively reaggregated . In the presence of fluoride no monomerization was seen, and the aggregation proceeded at a much faster rate . It is concluded that the fluorescence changes are not due to major relocalization of AlPc in the cells, but to light-induced monomerization followed by reaggregation. The protective effect of fluoride may be due to the enhanced aggregation rate, because aggregated dye molecules are photochemically inactive. Because D 2 0 affects neither the initial enhanced fluorescence in the absence of fluoride nor the rapid decrease in its presence it appears that 102 is not involved in the photodynamic reactions leading to these changes .

1 . Introduction Photodynamic therapy (PDT) of malignant solid tumours using Photofrin ® and red light is now in phase III clinical trials . Because of the shortcomings of Photofrin as a photosensitizer for PDT, secondgeneration sensitizers are being intensively studied . "Author for correspondence. Nuclear Research CenterNegev, PO Box 9001, Beer-Sheva 84190, Israel. tCenter for Bio-Pharmaceutical Sciences, and $Department of Medical Biochemistry, Sylvius Laboratory, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands .

Phthalocyanines (Pc) have shown promise, and some Pc derivatives are being considered for phase I clinical trials (for recent reviews see Van Lier 1990, Ben-Hur 1992, Rosenthal 1991) . For the rational use of a drug its molecular and cellular mechanisms of action have to be understood . PDT using Pc results in damage to plasma membrane, mitochondria and DNA (Ben-Hur et al. 1987a,b, Hunting et al . 1987, Ramakrishnan et al. 1988, Specht and Rodgers 1990) . The extent to which damage in each cellular structure contributes to cytotoxicity, however, is not known . Recently, fluoride has been shown to form a complex with AlPc derivatives, resulting in modified binding of the dye to proteins and a modified photodynamic action (Ben-Hur et al. 1991 a) . This results in protection by fluoride against AIPcS 4-induced photohaemolysis of human erythrocytes (Ben-Hur et al . 1991c) and A1Pc-induced photocytotoxicity in Chinese hamster cells (Ben-Hur et al . 1992a). In the former case this may be due to inhibition of crosslinking of spectrin monomers (Ben-Hur and Orenstein 1991) and in the latter to protection against photodynamic damage to the plasma membrane enzyme Na + /K + ATPase (Ben-Hur et al. 1992b) . The localization of Pc derivatives in cells, and changes occurring before and during illumination, can be followed using fluorescence microscopy (Ruck et al. 1990, Peng et al . 1990, Chan et al. 1989) . The aim of this work was to follow the changes in fluorescence of AlPc in CHO cells during illumination in the presence and absence of fluoride, in order to understand the mechanism of its protective action . The photostability of the dye was not affected by fluoride, but A1Pc was rapidly aggregated during illumination in its presence, while the opposite phenomenon occurred in its absence . The conversion of the dye into photochemically inactive aggregates may underlie the protective action of fluoride in CHO cells .

0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd

E. Ben-Hur et al .

768 2. Materials and methods 2.1 . Chemicals

A1Pc was obtained from Eastman Kodak, Rochester, NY . The dye was stored as a 1 mm stock solution in dimethylformamide . D 20 (99 . 9 atom% D) was from Aldrich . All other chemicals were analytical grade .

medium containing 3 µM AlPc. The cells were rinsed with DPBS and suspended by scraping into DPBS . Five millilitre cell suspensions (1 x 10 6 cells/ml) were illuminated as above . Absorption spectra were recorded at intervals using a spectrophotometer (Beckman DU-64) . The spectra shown were corrected for absorption of a cell suspension not containing dye .

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2 .6. Digital fluorescence microscopy 2.2 . Cell culture CHO epithelial cells, clone K, ATCC number CCL61, were grown attached to plastic Petri dishes in Ham's F10 medium supplemented with 15% heat-inactivated newborn calf serum in a humidified atmosphere containing 5% CO 2 at 37°C . 2.3 . Light exposure The light source was a slide projector equipped with a 150 W quartz halogen light bulb . The light was filtered by a cut-off filter (A> 605 nm) . The incident fluence rate was 200 W m -2 .

2 .4 .

Fluorescence measurements

Changes in fluorescence of AlPc-loaded CHO cells were measured after 2 h incubation in growth medium containing 1 µM A1Pc. Under these conditions the dye and solvent were not toxic in the dark . Exposure to light for 30 s resulted in 80% and 10% cell survival, in the presence and absence of fluoride, respectively . The cells were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and suspended by scraping into DPBS . Five millilitre cell suspensions (2 x 10 3 cells/ml) were illuminated in a beaker with continuous stirring . Fluorescence was measured at intervals during illumination, A =607 nm, ' em =680nm, in a spectrofluorimeter (Aminco SPF 500) . In some experiments the cells were extracted with dimethylformamide after various irradiation times and fluorescence was measured as above in the cell extract . For extraction the cells were pelleted and resuspended in 3 ml solvent . No A1Pc was detected in the DPBS supernatant . 2.5 . Absorption measurements Absorption spectra of the A1Pc-loaded CHO cells were measured after 2 h incubation in growth

Cells were grown on glass coverslips and incubated with 1 µM AlPc for 2 h . The cells were then rinsed and resuspended in 2 ml DPBS and illuminated for various times in the presence or absence of 5 mm NaF . The coverslips were placed into an incubation chamber, buffer was added on top, and the chamber mounted on a Zeiss 1 M 35 microscope with quartz optics . To obtain AlPc fluorescence from the cells, an excitation wavelength of 340 nm, a dichroic mirror (510 nm) and a long pass emission filter with a cut-off at 520 nm, were used . Exposure time, by computer-controlled shutters, was 3 s . Images were obtained with a Series 200 CCDcamera system (Photometrics, Tucson, AZ) and processed on an Imagine system (Synoptics, Cambridge, UK) . After background subtraction, the average intensity of the pixels of every individual cell was calculated . Photographs were taken from the screen. 3. Results Figure 1 shows the fluorescence changes that occurred in A1Pc-loaded cells during exposure to red light . The fluorescence initially increased, reaching a maximum at about 10 min. Continued illumination caused a progressive decrease in fluorescence . The photobiology of phthalocyanines depends on the presence of O 2 but the contribution of types I and II photodynamic reactions to the overall effect is still debated (Ben-Hur 1992) . D 20 is used as a test for singlet oxygen ( 102) involvement since the lifetime of 0 2 is about 10 times longer in deuterated solvents (Kajiwara and Kearns 1973) . Illumination of cells suspended in D 20 did not affect the initial rise in fluorescence but enhanced its subsequent decline, suggesting that 10 2 may be involved in the latter process . When exposure of the cells to light was in the presence of 5 mm NaF there was no initial enhancement of fluorescence . Instead, fluorescence declined very rapidly, at a rate 10-fold faster than in the absence of fluoride (Figure 1) . D 20 had no effect

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Phthalocyanine fluorescence in cells

on the rate of disappearance of fluorescence in the presence of fluoride. To test whether the decline in fluorescence was due to photodegradation of A1Pc or to its redistribution in the cell, the dye was extracted from the cells after various illumination times and its fluorescence measured . The results show that A1Pc in the cells was degraded to a small extent at a rate that was not dependent on the presence of fluoride (Figure 1) . Since photodegradation of A1Pc cannot explain the fluorescence change we used fluorescent microscopy to see whether the dye is redistributed in the cell during illumination . Figure 2a shows that prior to irradiation the dye was localized all over the cytoplasm in numerous organelles and bound to cellular membranes . Following light exposure this pattern of distribution was not changed, but the fluorescence of the cells decreased (Figure 2b) . The decrease in fluorescence was markedly evident after illumination in the presence of fluoride (Figures 2c and 2d) . However, no obvious change in overall dye distribution could be seen . Most phthalocyanines form stacked aggregates in the presence of water molecules . Aggregation is reflected in the absorption spectrum by hypso- and hypochromism (Darwent et al. 1982) . Figure 3 shows the changes in the absorption spectrum of AlPc in cells during illumination . Because of the lower sensitivity of absorbance measurements compared to fluorescence the amount of dye used was increased from 1 to 3 µM . The results obtained were indepen-

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LIGHT EXPOSURE (min) Figure 1 . Fluorescence changes of AIN in CHO cells loaded with 1 µM AIN for 2 h and then suspended in DPBS with (A) or without (0) 5 mm NaF in H20 and D2O (solid symbols) . The fluorescence of cell extracts exposed without (o) and with (∎) 5 mm NaF are also shown . Standard errors were smaller than the symbols .

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dent of the dye concentration in this range . The most striking change was the increased absorption of the main peak at 675 nm, reaching a maximum after 10 min light exposure . Additional illumination caused progressive reduction of the main peak and the apperance of a broad peak at 620-640 nm . When A1Pc-loaded cells were illuminated in the presence of fluoride there was no initial increase of the main peak . On the contrary, absorption at 675 nm rapidly decreased with concomitant increase around 640 nm (Figure 4) .

4. Discussion A1Pc is a hydrophobic dye which is waterinsoluble and binds tightly to various proteins (BenHur et al . 1991a) . When taken up by the cells it appears to localize in numerous cytoplasmic organelles and cellular membranes (Figure 2) . Because double-staining with organelle-specific fluorescing dye has not been done it is not certain in what organelles the AlPc is localized . They are probably mitochondria and endoplasmic reticulum, but lysosomes cannot be excluded . This assumption is supported by our observation that light activation of A1Pc causes an immediate transient increase of cytoplasmic free calcium (Ben-Hur et al. 1991b) which is released from an intracellular store (data not shown) . Endoplasmic reticulum and mitochondria are the main calcium stores in the cell . Fluorescent microscopy demonstrates that during irradiation there were no obvious changes in the intracellular distribution of A1Pc, but the fluorescence intensity decreased progressively (Figure 2) . However, the sensitizer may need some time to redistribute after light exposure (Berg et al . 1991) . This fact may influence the conclusions from Figures 2c and 2d, where the time of light exposure was short and the cells investigated immediately afterwards . The changes in fluorescence during light exposure were measured (Figure 1) . Evidently, there was only a small amount of photodegradation of A1Pc, in the presence and absence of fluoride . This cannot account for the decrease in fluorescence, although it caused an underestimation of the initial fluorescence enhancement . A satisfactory explanation for the changes in fluorescence can be offered upon examination of the changes in the absorption spectra of dye-loaded cells during illumination (Figures 3 and 4) . The initial enhancement of the main absorption peak at 675 nm is typical for monomerization of phthalocyanines, and is consistent with the suggestion that light exposure of aggregated phthalocyanines may result



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E. Ben-Hur et al .

Figure 2 . Fluorescence micrographs of CHO cells loaded with 1 /1M A1Pc for 2 h . A : Control, no light exposure. Fluorescence intensity taken as 100%±17 (n=10) . B : 45 min light . Fluorescence intensity 56%±13 of control (n=8) . C : 3 min light in the presence of 5 mm NaF. Fluorescence was 51% ± 16 (n= 5) . D : 10 min light with 5 mm NaF. Fluorescence was reduced to 21%±8 (n=8) . The presence of 5 mm NaF reduced fluorescence to 68% in control, unirradiated cells (not shown) ; n indicates the number of cells used for analysis .

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Absorption spectra of CHO cells incubated with 3µM AIPc . Cell suspensions in DPBS were illuminated for 0 min 5 min (--- ), 10 min (- -) and 45 min ( . . . . ) prior to measurement .



Phthalocyanine fluorescence in cells

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Figure 4.

Absorption spectra of CHO cells incubated with 3µx A1Pc. Cell suspensions in DPBS containing 5 mm NaF were illuminated for 0 min (-), 5 min (--- ), 10 min (--) prior to measurement .

in monomerization (see references in Harriman and Richoux 1980) . Subsequent reduction of the main peak and increased absorption at 620-640 nm indicates aggregation of dye molecules . In the presence of fluoride no evidence for monomerization was seen, and aggregation proceeded at a much faster rate, consistent with the kinetics of fluorescence changes (Figure 1) . Interestingly, only the photochemical damage leading to aggregation in the absence of fluoride may be mediated by a type II photodynamic reaction . This is inferred from the observation that only in this case did D 2 0 enhance the rate of fluorescence change (Figure 1) . Moan and Anholt (1990) have recently observed fast transient changes in A1PcS 2 fluorescence in tumours during PDT. Our measurements were made about 2 min after illumintion, a time at which these transient changes disappear, and thus are unlikely to contribute to the changes in A1Pc fluorescence reported in this paper. These results may offer an explanation for the protective effect of fluoride against AlPc-induced photocytotoxicity. Excitation of phthalocyanines aggregates by light does not initiate useful photochemistry since the major pathway of deactivation is the internal conversion to the ground state (Darwent et al. 1982) . The rapid aggregation of A1Pc during illumination in the presence of fluoride, therefore, converts the dye to a photochemically inactive state . It should be noted that A1Pc is in a more aggregated state in the cells when complexed with fluoride even before illumination . This is deduced from a 30% reduction in fluorescence upon addition of 5 mm NaF to dye-loaded cells (not shown) and from the absorp-

tion spectra (compare Figures 3 and 4) . The extent of change in aggregation of A1Pc before and during illumination in the presence of fluoride is most probably not uniform at all cellular sites. Thus, selective protection by fluoride was observed for the photodynamic inactivation of various membrane functions (Ben-Hur et al. 1992b) . Our previous observation that fluoride reduced the binding of AIPc derivatives to various proteins (Ben-Hur et al. 1991 a) is consistent with the present results that fluoride induced aggregation of cellbound AIPc . This is because aggregation of porphyrins reduces their binding affinity to proteins (Cohen and Margalit 1990) . Finally, it appears that the magnitude of fluorescence changes of phthalocyanines during illumintion depends on the cell line (Ruck et al . 1990) . This was shown only for A1PcS 4 in two cell lines, and no explanation was offered by the authors . It would seem worthwhile, therefore, to extend the present study to other cell lines and AlPc derivatives . Aclmowledgement This work was supported by a grant from the Netherlands Cancer Foundation (IKW 89-01) . References BEN-HUR, 1992, Basic photobiology and mechanisms of action of phthalocyanines. In : Photodynamic Therapy : Basic Prin-

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ciples and Clinical Applications . Edited by : T . J . Dougherty and B . W . Henderson, pp . 63-77 (Marcel Dekker, New York) . BEN-HUR, E. and ORENSTEIN, A ., 1991, The endothelium and red blood cells as potential targets in PDT-induced vascular stasis . International Journal of Radiation Biology,

60, 293-301 . BEN-HUR, E ., FUJIHARA, T ., SuzuKI, F . and ELKIND, M . M., 1987a, Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines . Photochemistry

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and Photobiology, 45, 227-230 . BEN-HUR, E ., GREEN, M ., PRAGER, A ., KOL, R . and ROSENTHAL, I ., 1987b, Phthalocyanine photosensitization of mammalian cells : biochemical and ultrastructural effects. Photochemistry and Photobiology, 46, 651-656 . E ., DUBBELMAN, T. M . A. R. and BEN-HUR, VAN STEVENINCK, J ., 1991a, The effect of fluoride on binding and photodynamic action of phthalocyanines with proteins . Photochemistry and Photobiology, 54,

703-707 . E ., DUBBELMAN, T. M . A. R. and BEN-HUR, VAN STEVENINCK, J ., 1991b, Phthalocyanine-induced photodynamic changes of cytoplasmic free calcium in

Chinese hamster cells . Photochemistry and Photobiology, 54, 163-166 . BEN-HUR, E ., FREUD, A ., CANFI, A . and LIVNE, A., 1991c, The role of glycolysis and univalent ions in phthalocyanine sensitized photohaemolysis of human erythrocytes . International Journal of Radiation Biology, 59, 797-806 . BEN-HUR, E ., CLAY, M . E ., Vicioso, E . F., ANTUNEZ, A . R., RIHTER, B. D., KENNEY, M . E . and OLEINICK, N. L ., 1992a, Protection by the fluoride ion against of phthalocyanine-induced photodynamic killing Chinese hamster cells . Photochemistry and Photobiology, 55, 231-237 . BEN-HUR, E ., DUBBELMAN, T. M . A . R . and VAN STEVENINCK, J ., 1992b, Effect of fluoride on inhibition of plasma membrane functions in Chinese hamster cells photosensitized by aluminum phthalocyanine . Radiation Research, (In press) . BERG, K ., MADSLIEN, K., BOMMER, J . C ., OFTEBRO, R., WINKELMAN, J . W . and MOAN, J ., 1991, Light induced relocalization of sulfonated meso-tetraphenylporphines in NHIK 3025 cells and effects of dose fractionates . Photo-

chemisty and Photobiology, 53, 203-210. CHAN, W . S ., MACROBERT, A . J ., PHILLIPS, D. and HART, I . R.,

1989, Use of charge coupled device camera for imaging of intracellular phthalocyanines . Photochemistry and Photobiology, 50, 617-624 . COHEN, S . and MARGALIT, R ., 1990, Binding of porphyrins to

human serum albumin . Structure-activity relationship . Biochemical Journal, 270, 325-330 . DARWENT, J . R ., DOUGLAS, P., HARRIMAN, A ., PORTER, G . and RIGHOUx, M . C ., 1982, Metal phthalocyanines and porphyrins as photosensitizers for reduction of water to hydrogen . Coordination Chemistry Review, 44, 83-126 . HARRIMAN, A . and RICHOUx, M . C ., 1980, Attempted photoproduction of hydrogen using sulfophthalocyanines as chromophores for three-component systems. Journal of the Chemical Society Faraday II, 76, 1618-1626 . HUNTING, D . J ., GowANs, B. J ., BRASSEUR, N . and VAN LIER, J . E ., 1987, DNA damage and repair following treatment of V79 cells with sulfonated phthalocyanines . Photochemistry and Photobiology, 45, 769-773 . KAJIWARA, T . and KEARNS, D . R ., 1973, Direct spectroscopic evidence for a deuterium solvent effect on the lifetime of singlet oxygen in water. Journal of the American Chemical Society, 95, 5886-5890 . MOAN, J . and ANHOLT, H ., 1990, Phthalocyanine fluorescence in tumors during PDT . Photochemistry and Photobiology, 51, 379-381 . PENG, Q., MOAN, J., NESLAND, J. M . and RIMINGTON, C., 1990, Aluminum phthalocyanines with asymmetrical lower sulfonation and with symmetrical higher sulfonation : a comparison of localizing and photosensitizing mechanism in human tumor lox xenografts . International Journal

of Cancer, 46, 719-726 . RAMAKRISHNAN, N ., CLAY, M . E ., XUE, L ., EVANS, H . H ., ANTUNEZ, A . R . and OLEINICK, N . L ., 1988, Induction of DNA protein crosslinks in Chinese hamster cells by the photodynamic action of chloroaluminum phthalocyanine and visible light . Photochemistry and Photobiology,

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HILDEBRANT, C ., KoLLNER, T., SCHNECKENBURGER, H . and STEINER, R ., 1990, Competition between photobleaching and fluorescence increase of photosensitizing porphyrins and tetrasulphonated chloroaluminum phthalocyanine . Journal of Photochemisty and Photobiology, B : Biology, 5, 311-319 . SPECHT, K . G . and RODGERS, M . A . J ., 1990, Depolarization of mouse myeloma cell membranes during photodynamic action . Photochemistry and Photobiology, 51, 319-324 . VAN LIER, J . E ., 1990, Phthalocyanines as sensitizers for photodynamic therapy of cancer. In : Photodynamic Therapy of Neoplastic Disease . Edited by : D. Kessel, vol. 1, pp . 279-290 (CRC Press, Boca Raton, FL) . RUCK,

The effect of fluoride on photodynamic-induced fluorescence changes of aluminium phthalocyanine in Chinese hamster cells.

Fluence-dependent changes in the fluorescence of aluminium phthalocyanine (AlPc) were measured in Chinese hamster ovary (CHO) cells using digital fluo...
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