Ultrastructural Changes in PAM Cells After Photodynamic Treatment with Delta-Aminolevulinic Acid-Induced Porphyrins or Photosan

Ultrastructural Changes in PAM Cells After Photodynamic Treatment with Delta-Aminolevulinic Acid-Induced Porphyrins or Photosan Sonja Radakovic-Fijan, Klemens Rappersberger,* Adrian Tanew, Herbert Ho¨nigsmann, and Bernhard Ortel† Division of Special and Environmental Dermatology and *Division of General Dermatology, University of Vienna, Austria; †Wellman Laboratories of Photomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.

Photodynamic therapy (PDT) is the combination of a photosensitizing drug (Ps) with light in the presence of oxygen leading to the generation of reactive molecular species and destruction of cancer cells. In this study we compared PDT with two Ps, the hematoporphyrin derivative Photosan (Ph) and delta-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX, with respect to mitochondrial function and ultrastrucural alterations. The effects of PDT were investigated in PAM 212 cells after different Ps incubation times, light doses, and post-treatment periods. Both Ps induced a light dose-dependent impairment of the mitochondrial function with the dose–response curve being steep for ALA and flat for Ph. The prolongation of the incubation time from 4 to 20 h resulted in an increased

reduction of mitochondrial activity after ALA PDT but not after Ph PDT. Treatment with an irradiation dose that decreased mitochondrial activity by 50% (IC50) led to early and profound changes of mitochondrial morphology in ALA photosensitized cells, whereas photosensitization with Ph resulted in more pronounced alterations of lysosomes. We conclude that at bioequivalent sublethal PDT exposures of PAM 212 cells, ALA-induced damage is primarily restricted to mitochondria, whereas Ph-induced cytotoxicity is mediated by damage of the lysosomal system. Key words: delta-aminolevulinic acid/mitochondrial activity/ photodynamic therapy/Photosan/ultrastructure. J Invest Dermatol 112:264–270, 1999

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metabolites, mainly protoporphyrin IX (PP IX) (Malik and Lugaci, 1987). Clinical studies have demonstrated the efficacy of ALA PDT in the treatment of basal cell carcinoma, Bowen’s disease, and solar keratoses (Kennedy and Pottier, 1992; Wolf et al, 1993; Fijan et al, 1995). In this study we applied two clinically relevant Ps and investigated the effects of PDT with ALA or Photosan (Ph) on mitochondrial function and ultrastructural morphology of PAM 212 cells under different experimental conditions. We have chosen the murine keratinocyte line PAM 212 because our prior work on ALAinduced PP IX formation and modification by iron complexation was performed in these cells (Ortel et al, 1993) and was applied clinically with consistent results (Fijan et al, 1995). Most importantly, we wanted to investigate the differential effects of two diverse photodynamic regimens on the cellular ultrastructure, but did not address differences between cell lines, which can be considerable (Iinuma et al, 1994; Ortel et al, 1998). MTT assays and electron microscopy were performed after two different Ps incubation times (4 and 20 h), increasing irradiation doses and variable post-irradiation periods (1 and 24 h). To analyze the effects of sublethal irradiations and to differentiate between ALA and Ph-specific subcellular targets, we determined the ultrastructural changes after PDT with a bioequvalent PDT dose that reduced the mitochondrial activity by 50% (IC50).

hotodynamic therapy (PDT) is a treatment modality that employs the combined administration of a photosensitizing drug (Ps) with subsequent light irradiation (Dougherty et al, 1990; Moore et al, 1997). Systemic or local administration results in accumulation and retention within malignant tissues. Irradiation of the tissue-localized Ps with light of appropriate wavelength leads to cell death and tumor necrosis (Henderson and Dougherty, 1992; Fisher et al, 1995). One of the most commonly used Ps is hematoporphyrin derivative (HpD), which is a mixture of monomeric and oligomeric forms of various hematoporphyrins. HpD is commercially available as Photofrin or Photosan, and is used in fluorescence diagnosis and PDT of various internal neoplasms. In dermatology, PDT with topically administered delta-aminolevulinic acid (ALA) has become an established and increasingly used treatment modality in recent years (Kennedy and Pottier, 1992). ALA is the first precursor in heme synthesis. The biosynthesis of ALA is regulated by a negative feedback control by heme of ALA-synthetase. By addition of exogenous ALA this negative feedback mechanism can be bypassed. This leads to an increased synthesis and accumulation of downstream

Manuscript received May 25, 1998; revised November 9, 1998; accepted for publication November 19, 1998. Reprint requests to: Dr. Sonja Radakovic-Fijan, Division of Special and Environmental Dermatology, Department of Dermatology, University of Vienna Medical School, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Abbreviations: ALA, aminolevulinic acid; HpD, hematoporphyrin derivative; PDT, photodynamic therapy; Ph, Photosan; PP IX, protoporphyrin IX; Ps, photosensitizing drug.

MATERIALS AND METHODS Cells PAM 212 is a clonogenic and tumorigenic cell line derived from murine epidermal keratinocytes. The cells were grown continuously in RPMI 1640 medium (Gibco, Paisley, Scotland), supplemented with 10%

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fetal calf serum, 100 µg streptomycin per ml, and 100 IU penicillin (Gibco) in a humidified 5% CO2 atmosphere.

identically with the exception that the peak of fluorescence intensity used for quantitation was at 626 nm.

Chemicals Delta-aminolevulinic acid hydrochloride (Fluka, Buchs, Switzerland) was dissolved to a final concentration of 1 mM in complete medium. Photosan (Seelab, Vienna, Austria) was diluted in phosphatebuffered saline (PBS, Gibco) and used at a final concentration of 7.5 µg per ml or 15 µg per ml in complete medium.

Photodynamic treatment Two 3 105 cells were plated into 60 mm culture dishes. Forty-eight hours later the medium was replaced by 2 ml of complete medium containing the Ps. To attain roughly equal intracellular levels of Ph, the concentration of 15 µg per ml was used for the incubation period of 4 h, whereas the lower concentration of 7.5 µg per ml was used for the incubation period of 20 h. ALA was used at a concentration of 1 mM for both incubation periods. After incubation with the Ps the medium was removed and the cells were rinsed twice with PBS, covered with 1 ml PBS, and exposed to 0.5–3.0 J per cm2 of 514 nm coherent light radiation at a fluence rate of 50 mW per cm2. After a further incubation period for 20 h in fresh complete medium the mitochondrial function was determined. The irradiation dose that reduced the mitochondrial activity by 50% was termed IC50. Untreated cells, cells incubated with ALA or Ph without light exposure, and unphotosensitized cells treated with laser light only served as control. The ultrastructural studies were performed in two sets of experiments. In the first set the cells were incubated with ALA or Ph for 4 or 20 h and irradiated with a constant dose of 3 J per cm2. After a post-treatment period of 1 or 24 h the samples were processed for electron microscopy. In the second series of experiments the cells were incubated with ALA or Ph for 4 h and irradiated with the IC50. The post-treatment periods were again 1 or 24 h.

Radiation source A Coherent Medical 920 Argon Dye Laser (Coherent, Cambridge, U.K.) was used at 514 nm in the continuous wave mode. The irradiance was measured at the level of the cells with a Model 210 power meter (Coherent). Quantitation of porphyrins The cellular content of PP IX and Ph was determined after an incubation period of 4 and 20 h. The cells were washed twice with PBS (without Ca211 and Mg211) and detached from the culture dishes using 1 ml of 0.1% trypsin/EDTA (Gibco). After dilution with 2 ml of cold PBS, 500 µl of the suspension was used for cell counting (Coulter Electronics, Luton, U.K.). The remaining 2.5 ml were centrifuged and the pellet was dissolved in 3 ml of 2% SDS in 0.1 M NaOH (NaOH/ SDS). Fluorescence was measured in a LS 50B spectrofluorometer (Perkin Elmer, Beaconsfield, U.K.). The excitation wavelength was set at 400 nm, the emission spectrum was recorded from 600 to 720 nm rather than at a single wavelength to allow correction for background scattering and to exclude contribution from non-PP IX porphyrins. The peak height at 631 nm was used for quantitation of PP IX according to a standard curve. The amount of PP IX was expressed as fg per cell. All these steps were done under minimal lighting conditions. Photosan was quantitated

Figure 1. Reduction of MTT conversion after photosensitization with ALA or Ph and irradiation with an Argon dye laser (514 nm). n, 1 mM ALA per 4 h; u, 1 mM ALA per 20 h; s, 15 µg Ph per ml per 4 h; e, 7.5 µg Ph per ml per 20 h. Values are given as mean 6SD.

MTT assay For the evalution of the mitochondrial function we slightly modified the MTT conversion assay (Mosmann, 1983), which measures mitochondrial dehydrogenase activity. Twenty hours after PDT the medium was removed and the cells were washed once with PBS at room temperature. One milliliter PBS containing 1.5 mg MTT [3-(4,5-dimethyl-thiaziol-2yl)-2,5-diphenyl tetrazolium bromide, Sigma] per ml was added and the cells were incubated at 37°C for 4 h. The supernatant was replaced by 2 ml dimethylsulfoxide (Merck, Vienna, Austria) and the dishes were shaken at a moderate speed for 30 min. The optical density of each solution was measured at 576 nm and the values of treated cells were expressed as percentage of untreated controls. Experiments were done in duplicates and repeated six times. Transmission electron microscopy Photosensitized cells were fixed in 2.5% glutaraldehyde for 5 h, washed in 0.1 M cacodylate at 4°C, postfixed in 3% osmium tetroxide for 1 h, washed in distilled water, and left in 0.5% uranylacetate-veronal buffer for 1 h at room temperature. The cells were then dehydrated in a graded series of ethanol and subsequently embedded in Epon 812. Thin sections were cut on a Reichert Ultracut 2000, stained with 1% uranyl acetate and 0.25% lead citrate, and examined with a transmission electron microscope (JEOL-EX 1200, Tokyo, Japan).

Table I. Synopsis of ultrastructural findings after different conditions of ALA and Ph PDT ALA PDT Light dose 3 J/cm2

IC50a

a

Incubation time (h)

Time after PDT (h)

4/20

1

4/20

24

4

1

4

24

Ph PDT Ultrastructural findings

Pronounced cytoplasmic edema Various degrees of mitochondrial swelling up to complete destruction Moderate swelling of the endoplasmatic reticulum Progression of cell damage cell death Slight to severe cytoplasmic edema Mitochondrial condensation and various degrees of hydropic swelling Occasionally swelling of the endoplasmatic reticulum Progression of cell damage Cell death

Irradiation dose leading to 50% reduction of mitochondrial activity.

Incubation time (h)

Time after PDT (h)

4/20

1

4/20

24

4

1

4

24

Ultrastructural findings Moderate cytoplasmic edema Large number of membrane bound cytoplasmic vesicles filled with granular material Discrete damage of mitochondria Progression of cell damage Cell death Moderate cytoplasmic edema Small number of cytoplasmic vesicles filled with granular material Occasionally mitochondrial swelling Large number of phagolysosomes

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RESULTS Similar cellular porphyrin levels after Ps incubation for 4 or 20 h Spectrofluorometry of extracts from cells treated with ALA showed a peak emission at 631 nm characteristic of PP IX. The cellular PP IX content after ALA incubation for 4 and 20 h was 193.5 6 24.2 fg per cell and 150.7 6 19.3 fg per cell, respectively (mean 6SD of six determinations). Control cells without ALA did not yield measurable amounts of intracellular PP IX. The fluorescence spectra from cells treated with Ph had a peak emission at 626 nm. The cellular content of Ph was 16.1 6 1.5 fg per cell after 4 h incubation with 15 µg Ph per ml and 19.8 6 2.1 fg per cell after 20 h incubation with 7.5 µg Ph per ml, respectively (mean 6SD of six determinations). Mitochondrial activity is severely impaired after ALA PDT Incubation of PAM cells with ALA or Ph and subsequent light irradiation resulted in a dose-dependent reduction of the mitochondrial function as measured by MTT conversion (Fig 1). The slopes of the dose response curves were, however, clearly different for the two Ps. With ALA PDT the slope was steep in contrast to a flat slope with Ph PDT. Treatment with ALA for 4 h and the lowest irradiation dose of 0.5 J per cm2 decreased the mitochondrial function by µ40%. After 20 h of ALA incubation the same light dose reduced the MTT converting ability by as much as 90%. Higher irradiation doses led to a progressive impairment of the mitochondrial function, which was completely abolished after both incubation periods and a dose of 3 J per cm2. The IC50 was 0.62 J per cm2 after an ALA incubation period of 4 h and 0.3 J per cm2 after ALA incubation for 20 h. The reduction of mitochondrial activity was less pronounced after Ph PDT and only slightly influenced by the Ph incubation time (Fig 1). The IC50 was 2.2 J per cm2 for the incubation period of 4 h and 2.0 J per cm2 after Ph incubation for 20 h, which was in accordance with the similar cellular porphyrin levels. Mitochondrial swelling is an early result of ALA PDT, whereas Ph PDT induces lysosomal damage after high-dose irradiation (Table I) Four or 20 h incubation with ALA followed by irradiation with 3 J per cm2 and a post-irradiation period of 1 h led to similar degrees of cellular damage. A pronounced edema of the cytoplasm was seen that was primarily confined to the periphery of the cells (Fig 2a). In addition, we detected a moderate swelling of the rough and smooth endoplasmatic reticulum and various degrees of hydropic swelling of the mitochondria, occasionally leading to complete destruction of these organelles (Fig 2b). In contrast, the Golgi apparatus, lysosomes, as well as cytoskeletal components such as keratin filaments and microtubules, appeared normal (Fig 2c). Twenty-four hours after ALA PDT a progression of these ultrastructural changes and signs of incipient karyolysis were detected. Cells exposed to Ph for 4 or 20 h and irradiated with 3 J per cm2 also disclosed similar ultrastructural changes 1 h after treatment. A moderate cytoplasmic edema was present with large numbers of individual ribosomes located at the periphery of the cytoplasm (Fig 3a). Between this cytoplasmic region and the nucleus, damaged lysosomes were found as membrane bound vesicles of different sizes filled with an electron dense granular material (Ghadially, 1988) (Fig 3b). These vesicles were never seen in Ph-treated unirradiated control cells or cells after ALA PDT. All other cell organelles were centralized and arranged around the nucleus. They Figure 2. Ultrastructural findings 1 h after ALA PDT with 3 J per cm2. (a) Pronounced edema (E) of the cytoplasm in the periphery of the cell; (b) various degrees of mitochondrial degeneration (M) with loss of mitochondrial cristae and swelling of the rough and smooth endoplasmatic reticulum (ER); (c) lysosomes (L) and keratin filaments (KF) appear normal. Scale bars: (a) 1 µm; (b, c) 0.2 µm.

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were morphologically well preserved with the exception of slightly condensed mitochondria and discrete swelling of the endoplasmatic reticulum (Fig 3c). Twenty-four hours after Ph PDT progression of the ultrastructural changes and true cell death occurred. Biologically comparable sublethal doses of ALA PDT and Ph PDT result in different ultrastructural response patterns (Table I) One hour after ALA PDT with the IC50 the cells displayed slight to moderate cytoplasmic edema (Fig 4a). The nucleus was normal and surrounded by a rim of regular appearing cytoplasm that contained most if not all cell organelles. At the periphery of the cells the cytoplasm contained ribosomes without evidence of other cell organelles. Again, the ultrastructural changes of the mitochondria were prominent with signs of condensation or hydropic swelling (Fig 4b). These changes, however, were less pronounced than after irradiation with 3 J per cm2. Occasionally, the cells had a foamy appearance due to masses of membrane bound vacuoles within the cytoplasm. Some of the vacuoles contained parts of mitochondrial cristae. Hydropic swelling of the endoplasmatic reticulum was also seen, but other subcellular structures such as the cell cytoskeleton and Golgi apparatus appeared unaffected. Individual cells with pronounced cytoplasmic changes also revealed margination of the chromatin indicative of early karyolysis. Twenty-four hours after ALA PDT these ultrastructural changes clearly progressed and were characterized by a marked cytoplasmic edema, pronounced swelling of the mitochondria and endoplasmatic reticulum, and signs of frank karyolysis (Figs 4c, d). One hour after Ph PDT with the IC50 we observed moderate cytoplasmic edema, many lipoidic vacuoles, and occasional condensation or swelling of the mitochondria. Around the nucleus a small number of altered lysosomes were present (Fig 5a). An overall similar morphology was found 24 h after Ph PDT with the notable exception of increased numbers of phagolysosomes (Fig 5b). DISCUSSION This study was designed to determine the differences in functional and ultrastructural changes after PDT with the exogenous HpD Ph or ALA-induced endogenous PP IX in the murine keratinocyte cell line PAM 212. In particular, we addressed the impact of Ps incubation time, irradiation dose, and time interval after PDT on the quantity and quality of ultrastructural alterations. The use of the IC50 allowed us to investigate the effects of sublethal irradiation and to compare the ultrastructural changes after equiphototoxic doses of ALA PDT and Ph PDT. ALA treatment resulted in high concentrations of PP IX after an incubation period of 4 or 20 h. The intracellular PP IX concentration was µ20% lower after ALA incubation for 20 h as compared with 4 h, which might be due to the presence of 10% fetal calf serum in the cell medium. The presence of special components in the serum with high affinity for porphyrins can cause the translocation of PP IX from the cells to the medium (Fukuda et al, 1993). Contrary to the kinetics of intracellular PP IX accumulation, ALA PDT-induced reduction of mitochondrial activity was greater after an incubation period of 20 h than after 4 h. This is in agreement with other studies that demonstrated increased phototoxicity with

Figure 3. Ultrastructural findings 1 h after Ph PDT with 3 J per cm2. (a) The periphery of the cytoplasm is filled with ribosomes (R). The nucleus (N) is localized in the center and surrounded by cell organelles and intermediate filaments. Numerous membrane-bound vesicles (V) of different sizes filled with electron dense granular material are found in the cytoplasm. (b) Higher magnification of the membrane-bound vesicles (V) that are partly or completely filled with granular electron dense material. (c) Discrete damage of mitochondria (M) and endoplasmatic reticulum (ER). Scale bars: (a) 1 µm; (b) 0.2 µm; (c) 0.5 µm.

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Figure 4. Ultrastructural findings after ALA PDT with the IC50. One hour after irradiation: (a) cytoplasmic edema (E) and mitochondrial condensation (arrow); (b) higher magnification of mitochondrial condensation (arrow). Twenty-four hours after irradiation: (c) progression of cell damage; marked edema of the cytoplasm (E), karyolysis (N), and (d) pronounced swelling of the mitochondria (M) and endoplasmatic reticulum (ER). Scale bars: (a, c) 1 µm; (b, d) 0.5 µm.

prolonged ALA incubation but failed to find a correlation with the intracellular PP IX levels (Iinuma et al, 1994; Gibson et al, 1997). The reason for the incubation time-dependent increase in reduction of mitochondrial activity might be that PP IX redistributes within the mitochondria, leading to PDT-induced inhibition of differently located mitochondrial enzymes (Gibson et al, 1989). Incubation with 7.5 µg Ph per ml for 20 h resulted in µ20% higher intracellular concentrations of Ph than incubation with 15 µg per ml for 4 h. The effects on mitochondrial activity correlated with the intracellular Ph levels at all except the highest irradiation doses and were substantially lower than those observed after ALA PDT. These findings indicate that contrary to ALA PDT the mitochondria were not the primary target of Ph photosensitization under our experimental conditions. This is in contrast to other studies with the related HpD compound Photofrin, in which

mitochondrial damage was the predominant ultrastructural finding after PDT (Coppola et al, 1980; Milanesi et al, 1989). In this regard, besides the delivery system (Malik and Faraggi, 1992) the cell line may have a major role as different subcellular Ps localizations and Ps-induced mechanisms of cellular damage were observed in different cell lines (Berg and Moan, 1997). ALA PDT with the high irradiation dose of 3 J per cm2 led to early swelling of the mitochondria followed by swelling of the endoplasmatic reticulum and a dramatic progressive disintegration of the cells. ALA-induced PP IX is synthetized in the mitochondria and has a high affinity to membranes due to its pronounced hydrophobicity. It is thus assumed that time-dependent translocation and accumulation of PP IX in cell organelles other than the mitochondria results in their photodestruction (Malik and Lugaci, 1987; Malik et al, 1989; Gaullier et al, 1995). This notion is

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Figure 5. Ultrastructural findings after Ph PDT with the IC50. One hour after irradiation: (a) mild cytoplasmic edema (E), many lipoidic vacuoles (LV), and cytoplasmic vesicles filled with granular material (V). Twenty-four hours after irradiation: (b) numerous phagolysosomes (PL), vesicles with granular content (V), and condensed mitochondria (M). Scale bars: (a) 1 µm; (b) 0.5 µm.

supported by fluorescence studies demonstrating a typical mitochondrial pattern after short incubation with ALA (Iinuma et al, 1994; Liang et al, 1998) in contrast to a more diffuse pattern with large perinuclear spots after ALA incubation for 42 h (Gaullier et al, 1995). In our study we found that the morphologic changes after ALA PDT were largely independent of the ALA incubation time but increased with the longer post-treatment period. This indicates that a 4 h ALA incubation period is sufficient for maximum photosensitization, whereas a 1 h post-treatment time is too short for the full development of ultrastructural changes. Besides direct

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photochemical reactions, PDT-mediated cell damage is supposed to involve enzymatic processes that require a longer time in exerting their effects (He et al, 1994). After Ph PDT with the high irradiation dose of 3 J per cm2 and a post-treatment interval of 1 h we observed large numbers of damaged lysosomes. The mitochondria showed only discrete damage and the other cell organelles were well preserved. Twenty-four hours after irradiation with 3 J per cm2 we found progression of the ultrastructural changes and signs of true cell death. Several indications are found in the literature that lysosomes might be a likely target for PDT using a HpD. Due to its mostly negative charge HpD is difficult to concentrate in the mitochondria but likely to accumulate in other parts of the cell. This notion is consonant with the findings of fluorometric studies that showed the absence of a mitochondrial fluorescence pattern after HpD treatment (Bottiroli et al, 1992; Krammer et al, 1993). Incubation of cells with HpD in serum-containing culture medium, as was done in our experiments, promoted the delivery and accumulation of porphyrins in lysosomes (Malik and Faraggi, 1992; Ge`ze et al, 1993). This conceivably renders the lysosomes a primary target for HpD-induced photodynamic reactions. Further support for the pivotal role of lysosomes comes from our observation that the lysosomal changes after Ph PDT are morphologically similar to those reported after PDT using Nile blue as photosensitizer, which was shown to localize in lysosomes (Lin et al, 1993). Irradiation of ALA-photosensitized cells with the IC50 resulted in cellular alterations that were qualitatively similar albeit more discrete than after irradiation with 3 J per cm2; however, with the lower irradiation dose the degree of cellular damage was much more variable between single cells as compared with high dosetreated cells where the cell damage was rather uniform. In addition, a small fraction of IC50-irradiated cells appeared unaffected by ALAphotosensitization and showed no or only minimal ultrastructural alterations. This may reflect variable levels of ALA-induced PPIX within a subset of cells. As was the case with ALA the irradiation of Ph-photosensitized cells with the IC50 led to subcellular changes that were more discrete but otherwise identical to those seen after irradiation with 3 J per cm2. The prevailing finding 1 h after treatment with the IC50 was a small number of cytoplasmic vesicles filled with granular material that represented damaged lysosomes. After the posttreatment period of 24 h in addition to these vesicles a large number of phagolysosomes was observed, reflecting increased lysosomal activity and induction of autophagocytosis. This indicates the activation of a reparative process that sequesters and degrades damaged parts of the cell (Zdolsek et al, 1990). Summarizing our data we conclude that at least in PAM 212 cells, (i) incubation with ALA results in marked and incubation time-dependent photosensitization of the mitochondria, (ii) the effect of Ph PDT on mitochondrial activity is much weaker than that of ALA PDT and only slightly affected by the Ph incubation time, (iii) the functional effects of ALA PDT are paralleled by pronounced and predominant alterations of mitochondrial morphology, and (iv) Ph PDT targets the lysosomal system and induces autophagocytosis.

This work was supported by a grant from the ‘‘Fonds zur Fo¨rderung der Wissenschaftlichen Forschung’’ (P 8532 MeD), Vienna, Austria.

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