Photochemistry and Photobiology Val. 46. No. 5. pp. 645-649, 1987 Printed in Great Britain. All rights reserved

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Section I11 - Biochemical and Subcellular Photobiology

MITOCHONDRIAL PHOTOSENSITIZATION BY PHOTOFRIN I1

GURMIT SINGH*, W. PATRICK JEEVES, BRIAN C. WILSON and DANIEL JANG Ontario Cancer Treatment and Research Foundation, and McMaster University. Hamilton, Canada

(Received 28 May 1987; accepted 1 June 1987)

Abstract-V-79 Chinese hamster cells grown as monolayers or as multicell spheroids were treated with Photofrin I1 (10 pg me-l for 16 h) and various doses of red light irradiation. The resulting biochemical and functional damage to cell mitochondria was studied. The activities of both succinic dehydrogenese and cytochrome c oxidase were found to decrease in a light dose-dependent manner. The respiratory control quotient (RC) decreased in parallel with a decrease in the activities of the respiratory chain proteins. Our data also showed a distinct temporal difference in the relative progression of rnitochon- drial damage and cell death as assessed by loss of discrete Rhodamine-123 (Rh-123) localization and trypan blue infiltration, respectively. Mitochondrial damage was detected immediately, as seen by delocalization of Rh-123 resulting from dissipation of the electrochemical gradient in damaged mito- chondria. Trypan blue infiltration occurs with a distinct time lag. These findings are consistent with the hypothesis that, at least for long Photofrin 11 incubation times. the mitochondrion is a primary target of photosensitization. The subsequent changes in cell membrane permeability may be a delayed result of decreased bioenergetics of the Photofrin I1 photosensitized cell.

INTRODUCTION

In recent years, photodynamic therapy (PDT)? has shown much promise as an effective alternative treatment modality for a variety of localized tumors including those of the skin, lung, breast, bladder, and brain (Dougherty et al., 1985; Wilson and Jeeves, 1987). Although tumor cell photocytotoxic- ity is believed to result from the production of highly reactive intermediate singlet oxygen (Dougherty et al., 1976), relatively little is known concerning either the basic mechanism of tumor cell destruction by PDT, or the critical subcellular site(s) of damage. However, considerable evidence has recently accumulated to suggest strongly that mitochondria are an important subcellular site of PDT damage, resulting in inhibition of cellular respiration (Gibson and Hilf, 1985; Atlante et al., 1986).

In this report, we examine the effect of Photofrin I1 (Photomedica, Raritan, NJ) on mitochondria1 damage of V-79 Chinese hamster cells grown as monolayer or as multicell spheroids utilizing bio- chemical and functional techniques. Biochemical damage was measured by a decrease in the activity of two enzymes, succinic dehydrogenase and cyto- chrome c oxidase. Mitochondrial function was moni- tored by (a) respiration and (b) the ability of mitochondria to concentrate Rh-123 via their elec- trochemical gradient. We used phase-contrast and fluorescence microscopy to investigate the relative progression of post-PDT loss of mitochondria, com- pared to cell viability. Delocalization of Rh-123, a

*To whom correspondence should be addressed. ?Abbreviations: HPD, hematoporphyrin derivative;

PDT, photodynamic therapy; MTT, 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide; TMPD, tetra- methyl phenylene diamine.

fluorescent dye specifically concentrated by viable mitochondria (Bernal et al., 1982; Shea et al., 1986) was used to follow the time course of loss of mito- chondrial viability, while trypan blue was used to assess plasma membrane viability.

MATERIALS AND METHODS

V-79 Chinese hamster cells were grown either as monolayers or as multicell spheroids in growth medium containing either 10% or 5% fetal bovine serum (Gibco, New York), respectively. The cells or the spheroids were treated with Photofrin I1 (Photomedica, Raritan, NJ) (10 pg m P i ) for 16 h, and washed in PBS, and subsequently irradiated with various fluences of red light. Irradiation was carried out using a bank of fluorescent bulbs (Type TLi83, Philips), filtered with red acetate cut-off filters (Roscolux No. 19, Rosco, CA) to give wide band illumi- nation above A = 585 nm. The fluence rate to which cultures were exposed was 9.22 W m - z in the range 623-634 nm which constituted 12.1% of the total filtered output from the source.

Succinic dehydrogenase assay. Succinic dehydrogenase in cells was measured using a modified colorimetric method of Mossman (Mossman et a l . , 1983). In this, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- mide (MTT) (Sigma Chemical Co. , St. Louis, MO) a pale yellow substrate, is converted to a dark blue formazan product by succinic dehydrogenase.

Cytochrome c oxidase assay. Cytochrome c oxidase was assayed polarographically by measuring the rate at which oxygen is consumed during the oxidation of cytochrome c . In this assay, a catalytic concentration of cytochrome c is kept in a reduced form by the presence of excess ascorb- ate and a small amount of tetramethyl phenylene diamine (TMPD) which is a redox dye that mediates the reduction of cytochrome c by ascorbate.

Mitochondrial respiration. Respiration in the cells was assayed polarographically using a Clark microelectrode (bore size 0.5 cm) at 30°C in a medium containing 0.225 M sucrose, 15 mM KCI, 1 mM EDTA, 5 rnM MgCL, 10 mM phosphate buffer and 50 mM Tris-HCl buffer, pH 7.4. Plasma membrane was permeabilized using digitonin as detergent and succinate (10 mM) was used as substrate.

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Figure 1. Effect of Photofrin I1 on mitochondrial respir- ation in V-79 cells incubated in 10 pg m t - ' of Photofrin I1 for 16 h and subsequently irradiated with various red light fluences (1.11. 2.78, 5.55, and 16.7 x 10' J m-* in the wavelength band 623-634 nm). Each value is the mean (? SEM) of 3-6 experiments. Each control value is an average of nine experiments with three different controls, i.e. (a) control-no drug and no light, (b) control-drug 10 pg m t - ' but no light. (c) control-no drug but red light (2.78 X 10' J m-*). The different controls, (a), (b)

and (c). were all very similar.

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Figure 2. Effect ot Photofrin I1 on cytochrome c-oxidase activity in V-79 incubated in 10 pg me-' of Photofrin I1 for 16 h prior to light irradiation with various light flu- ences. Each value is the mean (? SEM) of three individual experiments. Each control value is an average of nine experiments with three different controls, i.e. (a) control-no drug and no light, (b) c o n t r o l d r u g 10 pg me-- ' but no light, (c) control-no drug but red light (2.78 x 10' J m-z). The different controls, (a), (b) and (c),

were all very similar.

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111

Figure 3. Effect of Photofrin I1 on succinic dehydrogen- ase. V-79 cells (1 x loh cells m6-I) were incubated in 10 pg ,me- ' Photofrin I1 for 16 h prior to irradiation with various light fluences as shown. Each value is the mean (? SEM) of six experiments. Each control value is an average of nine experiments with three different controls, i.e. (a) control-no drug and no light, (b) control-drug 10 pg me-l but no light, (c) control-no drug but red light (2.78 x 10' J m-*). The different controls, (a), (b)

and (c), were all very similar.

Rhodamine-I23 juorescence. Cells or spheroids were incubated with Rhodamine-123 (5 pg mt- ' ) for 1 h at 37°C and washed with PBS prior to visualization. Stained cells were examined by epifluorescent illumination at 546 nm excitation. Photographs were made by using Kodak Ektachrome (ASA 400) film with the automatic exposure control of the microscope set at ASA 6300. Following Rhodamine-123 visualization, trypan blue was added to the medium in order to monitor cell membrane integrity.

RESULTS

Effect on mitochondria1 bioenergetics

Light irradiation of Photofrin 11-incubated V-79 cells led to a significant light-dose dependent depression of respiration (Fig. 1). The decrease in respiration could not be attributed to blockade in any specific complex of the respiratory chain. A light-dose dependent decreases in the activity of cytochrome c-oxidase was observed (Fig. 2), and a similar profile was obtained for succinic dehydro- genase (Fig. 3). These results indicate that Photofrin I1 photosensitization does not occur on a particular respiratory complex but could be generalized dam- age to the mitochondrial membrane which supports the various respiratory chain complexes.

Delocalization of Rh-123

In V-79 Chinese hamster cells exposed to 2.78 x J m-* red light (i.e. within the wavelength band

Mitochondria1 photosensitization 647

Figure 4. Fluorescence micrographs of V-79 cells incubated with 5 pg m l - ' Rh-123 for 30 min at 37°C prior to visualization. (a) Control: Untreated. Note the discrete localization o f dye. (b) Treated. (10 pg me- ] Photofrin I1 for 16 h + 2.78 x 10' J m-2 red light.) Most cells have lost their discrete concentration of Rhodamine-123 and a diffuse dull cytoplasmic fluorescence from Rhodamine-123 is observed. However, trypan blue is still excluded indicating cell membrane integrity. (- 2.5 p) .

623-634 nm) after prolonged incubation (16 h) in Photofrin I1 (10 kg m-'), results in the dissipation of the electrochemical gradient in mitochondria. This was observed by loss of specific mitochon- drially-localized Rh-123 fluorescence (Fig. 4a) which, after PDT treatment, diffusely filled with cytoplasm (Fig. 4b); nuclear sparing was still faintly observed. At this stage, trypan blue was excluded from the cells indicating that the cells retained an intact plasma membrane. Multicell spheroids tre- ated with Photofrin I1 and light also displayed a

similar temporal relationship between loss of mito- chondrial vs plasma membrane integrity. Cells in the spheroid which displayed Rh-123 fluorescence did not allow infiltration of trypan blue, but cells that did not fluoresce were trypan blue stained (Figs. 5a, b).

DISCUSSION

Photocytotoxicity by Photofrin I1 is believed to result from the production of highly reactive inter-

648 GURMIT SINGH et al.

Figure 5. (a) Fluorescence micrograph of treated 200 pm V-79 multicell spheroid (10 pg me ' Photofrin I1 for 16 h + 5.55 X lo3 J m-> of red light), incubated with 5 pg m t ' Rhodamine-123 for I h at 37°C prior to visualization. Only cells with viable mitochondria showed Rh-123 fluorescence (example shown by white arrow). (b) White light micrograph of treated V-79 multicell spheroid as for 5a. Following Rhodamine-123 visualization, trypan blue was added to the mcdiurn in order to detect cell membrane integrity. Most cells are stained with trypan blue except the cells that showed

fluorescence in 5a with Rhodamine-123 (e.g. white arrow). (- 25 pm) .

mediate singlet molecular oxygen (Dougherty et a/ . , times greater (Keene et ul., 1986). These photo- 1985). Photofrin I1 is aggregated in aqueous medium physical properties would suggest that the target of and is, therefore, a poor photosensitizer; however, Photofrin I1 would be the membrane. Since the in membranes it dissociates and the quantum yield cell has various membranes, such as the plasma of singlet oxygen formation becomes at least ten membrane, mitochondria1 membranes, lysosomal

Mitochondria1 photosensitization 649

membranes, nuclear envelope, endoplasmic retic- ulum and other organelle membranes, it is conceiv- able that one or a number of these membranes could be damaged resulting in photocytoxicity.

Various studies have demonstrated that the sub- cellular loci of hematoporphyrin derivative (HPD) photodamage depends to a large extent on the length of time that cells are exposed t o HPD prior t o irradiation (Moan et al., 1984; Kessel, 1984). For short incubation periods, plasma membrane is probably an important site of damage (Christensen et al., 1983). Prolonged incubation of cells with Photofrin 11, tends to localize it in mitochondria as shown by fluorescence microscopy (Berns et al., 1982) and by subcellular fractionation studies (Hisa- zumi et al., 1984). A number of mitochondrial mem- brane-associated enzymes are affected by Photofrin I1 (Hilf et al., 1984; Atlante et al . , 1986).

In this study, we confirm that cytochrome c oxi- dase and succinic dehydrogenase activities are reduced by Photofrin I1 photosensitization. This effect is also reflected in decreased respiration of photosensitized V-79 cells. It is evident that various mitochondrial membrane-associated proteins are photosensitized and thus the effect of Photofrin I1 is not associated with a single protein.

W e have demonstrated also, by the use of phase- contrast and fluorescence microscopy, that , for rela- tively prolonged Photofrin I1 incubation time (16 h), loss of plasma membrane integrity occurs after loss of mitochondrial viability. This temporal relationship between mitochondrial damage and plasma membrane integrity appears to decrease with increasing light dose. V-79 spheroids also indicate a correlation between mitochondrial damage and subsequent cell death. O u r findings are consistent with the hypothesis that mitochondria constitute an important, if not a primary target of Photofrin I1 photosensitization, a t least for long Photofrin I1 incubation times such as those relevant t o photody- namic therapy in vivo.

Acknowledgements-This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada.

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