ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 294, No. 1, April, pp. 30-37, 1992

Photodynamic Effects of Chloroaluminum Phthalocyanine Tetrasulfonate Are Mediated by Singlet Oxygen: ln l&o and in vitro Studies Utilizing Hepatic Microsomes as a Model Membrane Source’

Rajesh Agarwal, Syed I. A. Zaidi, Mohammad Athar,2 David R. Bickers, and Hasan Mukhtar3 Department of Dermatology, Skin Diseases Research Center, University Hospitals of Cleveland, Case Western Reserve University, and Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106

Received August 15, 1991, and in revised form November 11,199l

Chloroaluminum phthalocyanine tetrasulfonate (Al- PcTS) is a promising photosensitixer for the photodynamic therapy (PDT) of cancer. In this study, we investigated the in vivo and in vitro photodestruction of hepatic mi- crosomal membranes by AlPcTS and studied the role of reactive oxygen species in this process. Irradiation of he- patic microsomes prepared from AlPcTS-pretreated SENCAR mice to -6’75 nm light resulted in rapid de- struction of cytochrome P450 and associated monooxy- genase activities, and enhancement of lipid peroxidation in a light-dose-dependent manner. The specificity of AlPcTS and light dependency on photodestruction of mi- crosomal membranes was confirmed by Western blot analysis. Similar results were obtained when AlPcTS was added in vitro to a suspension of hepatic microsomes pre- pared from control animals followed by irradiation to -675 nm light. Among the quenchers of singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl rad- ical, only the quenchers of singlet oxygen such as sodium axide, histidine, and 2,5dimethyl furan afforded sub- stantial protection in a dose-dependent manner against AlPcTS-mediated photodestruction of cytochrome P450 and associated monooxygenase activities, and photoen- hancement of lipid peroxidation under both in vivo and in vitro conditions. These results suggest that lipid-rich mi- crosomal membranes may be the potential targets of cell injury by AlPcTS-based PDT and that this process is me- diated by singlet oxygen. o issz A&& pre~e. IUC.

r This work was supported by USPHS Grants CA 51602, ES-1900, POl-CA 48735, and P-30-AR-39750 and by research funds from the Department of Veterans Affairs.

’ Present address: Department of Medical Elementology and Toxi- cology, Hamdard University, New Delhi, India.

‘To whom correspondence should be addressed at Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44106.

30

Because of preferential retention of certain photosen- sitizers in malignant tissues compared to normal tissues, they are currently under intense study as an approach to the diagnosis and management of various human neo- plasms in a modality known as photodynamic therapy (PDT)4 of cancer (l-4). The photosensitizers initially employed for this purpose are porphyrins, Photofrin-I and Photofrin-II (4-6). Porphyrin-catalyzed photodamage has been shown to occur in various subcellular organelles in- cluding nuclei (7), mitochondria (a), lysosomes (9), and microsomes (10, 11). Our laboratory has demonstrated that photosensitization with porphyrins leads to the dis- ruption of membrane-bound enzymes, enhances the ca- tabolism of heme, and elicits peroxidative damage to lipid- rich membrane (10-14). The critical importance of oxygen in the PDT of cancer is confirmed by the fact that reactive oxygen species (ROS) play an important role in the pho- tocatalytic disruption of malignant cells (15). It is appre- ciated that the common denominator for membrane damage evoking cellular injury leading to tumor necrosis and cell death is the ability of the photosensitizers to produce ROS on exposure to appropriate wavelength of light (16). This has been confirmed by several in vitro and in uiuo experiments (15-19). The biological significance of ROS in PDT varies depending on the type of the pho- tosensitizer and its intracellular localization. Although evidence for the involvement of a particular ROS in the photodynamic action of porphyrins is not directly avail-

4 Abbreviations used: PDT, photodynamic therapy; AlPcTS, chlo- roaluminum phthalocyanine tetrasulfonate; PB, phenobarbital, AHH, aryl hydrocarbon hydroxylase, BP, benzo[a]pyrene; 3-OH-BP, 3-hy- droxybenzo[a]pyrene; ECD, 7-ethoxycoumarin-0-deethylase; ERD, 7- ethoxyresorufin-0-deethylase; MDA, malondialdehyde; 2,5-DMF, 2,5- dimethylfuran; P450, cytochrome P450; CO, carbon monoxide, SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ROS, reactive oxygen species; DTT, dithiothreitol; SOD, superoxide dismutase; ip, intraperitoneal.

0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc.

All rights of reproduction in any form reserved.

SINGLET OXYGEN-MEDIATED PHOTODYNAMIC EFFECTS 33

able, some reports suggest that porphyrins can interact with visible light by both Type I and Type II photochem- ical pathways (20, 21). Singlet oxygen generated via the Type II mechanism is thought to initiate most of the damage induced by PDT; the electrophilic nature of this ROS makes it efficient in producing oxidized forms of bio-molecules (22, 23). Though Type I reactions play a minor role in PDT due to competition between substrates and oxygen for the triplet state photosensitizer, they may be directly involved in cell damage especially when a pho- tosensitizer is bound or associated with easily oxidized biomolecules (4, 24).

While porphyrins are useful for the PDT of cancer, they do have a number of drawbacks related to their pharmacodynamics (4). These compounds bind to matrix proteins and are released very slowly over a matter of weeks, rendering the patients highly photosensitive for extended periods (3, 4, 16). This has led to a search for new photosensitizers for their use in PDT.

The phthalocyanines are a new class of photosensitizers which are extensively studied in recent years as promising alternatives to porphyrins in the PDT of tumors (25,26). Several studies utilizing in vitro cellular systems have shown that phthalocyanines are efficient photosensitizers (26-28) and produce very little, if any, photosensitization by room light (29). Of all the phthalocyanines known to date, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS) is the most studied photosensitizer for the PDT of tumors (26).

Although AlPcTS possesses significant photodynamic effects (29-32), the exact mechanism by which it evokes phototoxic injury is not well defined. In the present study we investigated the in vivo and in vitro photodestruction of hepatic microsomal membranes by AlPcTS and define the species of reactive oxygen responsible for photody- namic effect.

MATERIALS AND METHODS

Chemicals. AlPcTS was purchased from Porphyrin Prcducts (Logan, UT). Thiobarbituric acid, sodium azide, histidine, 2,5dimethylfuran (2,5-DMF), sodium benzoate, mannitol, superoxide dismutase (SOD), NADPH, resort&, and catalase were obtained from Sigma Chemical Co. (St. Louis, MO). Benzo[a]pyrene (BP) and 7-ethoxycoumarin were purchased from Aldrich Chemical Co. (Milwaukee, WI). 3-Hydroxy- benzo[a]pyrene (3-OH-BP) was provided by the Cancer Research Pro- gram of the National Cancer Institute, Division of Cancer Cause and Prevention (Bethesda, MD). 7-Ethoxyresorufin was a product of Pierce Chemical Co (Rockford, IL). All chemicals and equipments for electro- phoresis were from Bio-Rad (Rockville Center, NY). Nitrocellulose paper (0.45 pm) was from Schleicher & Schuell (Keene, NH), and other im- munochemical reagents were from KPIC (Bethesda, MD). All other chemicals were of highest purity commercially available.

Treatment of animals and preparation of hepotic microsomes. Hepatic microsomes were prepared from 6- to 8-week-old female SENCAR mice, obtained from the National Cancer Institute-Frederick Cancer Re- search Facility, Bethesda, MD. The animals were injected intraperito- neally (ip) with AlPcTS (5 mg/kg body weight in 0.2 ml of normal saline) or 0.2 ml of normal saline and were killed 24 h later and hepatic microsomes were prepared as described earlier (33). To obtain hepatic microsomes from rats pretreated with phenobarbital (PB), a standard

procedure published recently was employed (34). A microsomal pellet was suspended in 0.1 M potassium phosphate buffer, pH 7.4, containing 10 mM dithiothreitol (DTT), 100 pM MgClz, and 20% (v/v) glycerol and the aliquots were frozen at -170°C under liquid nitrogen. To protect against possible destruction of hepatic microsomes obtained from ani- mals pretreated with AlPcTS by room light, the entire preparative pro- cedure was conducted in the dark and all the glassware were covered with aluminum foil. The protein concentration was determined by the method of Bradford (35) using bovine serum albumin as standard.

Irradiation. Microsomal suspensions (0.5 to 2.0 mg protein/ml), in a final volume of 1 ml, were irradiated using a metal halide lamp equipped with a red filter. The emission peak of this light source, monitored by an IL-700 research radiometer (International Light, Newburg-Port, MA), was found to be a675 nm. The samples were placed in a petri dish surrounded by crushed ice and exposed to a desired dose of light fluence (J/cm’). Except in the case of AlPcTS-pretreated mouse hepatic mi- crosomes, desired amounts of AlPcTS were added in vitro to hepatic microsomal suspensions, before irradiation to ;~675 nm light. Each quencher was dissolved in 0.1 M potassium phosphate buffer, pH 7.4, and added immediately prior to irradiation, wherever needed.

Spectral analysis of hepatic microsomnl cytochrome P450 (P450). Hepatic microsomes (0.5 to 2.0 mg protein/ml) obtained from AlPcTS- pretreated mice were irradiated to various doses of light fluence (1 to 25 J/cm*), and carbon monoxide (CO) oxidized and sodium dithionite reduced minus sodium dithionite reduced difference spectra were re- corded using an Aminco DW-2a dual-beam spectrophotometer as de- scribed by Omura and Sato (36). In other experiments, hepatic micro- somes (0.5 to 2.0 mg protein/ml) obtained from control animals were added with various concentrations of AlPcTS (l-10 pg/ml) followed by irradiation (10 J/cm’) or were added with 2 pg/ml AlPcTS followed by irradiation to various doses of light fluence (l-25 J/cm’) and P450 spectra were recorded similarly. P450 content was determined using a molar extinction coefficient of 91 cm-’ mM-’ (36).

Western blot analysis of PB-pretreated rat hepatic microsomal P450. Hepatic microsomes (0.5 to 2.0 mg protein/ml) obtained from PB-pretreated rats were added with various concentrations of AlPcTS (0.5-12 fig/ml) followed by irradiation (10 J/cm’) or were added with 2 pg/ml AlPcTS and irradiated to various doses of light fluence (l-30 J/ cm*). These samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by the procedure of Laemmli (37) using 12.5% acrylamide gels in the presence of 0.1% SDS and 0.1% 2-mercaptoethanol. The resolved proteins were electropbo- retically transferred onto the nitrocellulose membranes according to the method of Towbin et al. (38) and probed, after blocking the nonspecific sites with 3% nonfat dry milk, by their sequential treatment with mono- clonal antibody 2-66-3 (a kind gift from Drs. S. S. Park and H. V. Gelboin, National Cancer Institute, Bethesda, MD) directed against purified rat liver P4502B1(39) and anti-mouse goat IgG coupled with alkaline phos- phatase. The color was visualized as described earlier (40).

Enzyme assays. Aryl hydrocarbon hydroxylase (AHH) activity was determined by a modification of the method of Nebert and Gelboin (41), as described earlier (34). 7-Ethoxycoumarin-0-deethylase (ECD) activity was determined according to a slight modification of the procedure of Greenlee and Poland (42), the details of which are described earlier (43). 7-Ethoxyresorufin-0-deethylase (ERD) activity was determined ac- cording to the procedure of Pohl and Fouts (44) as described earlier (34). The generation of malondialdehyde (MDA) was employed as a marker of lipid peroxidation and estimated by the method of Wright et al. (45) as described earlier (14,19). The effect of various quenchers was assessed by adding them to the reaction mixtures immediately prior to irradiation.

RESULTS

In Vivo Studies

As shown in Fig. 1, irradiation of hepatic microsomes obtained from AlPcTS-pretreated animals to various

32 AGARWAL ET AL.

I I I I I I I I I 410 420 430 440 450 460 470 460 490

Wavelength (nm)

FIG. 1. Light-fluence-dependent in vivo photodestruction of P450 in hepatic microsomes obtained from AlPcTS-pretreated SENCAR mice. Microsomes were suspended in 0.1 M phosphate buffer, pH 7.4, and irradiated to various doses of a675 nm light (1 to 25 J/cm’), and P450 spectra were recorded. Spectrum 1, unirradiated hepatic microsomes kept in the dark; spectra 2-8, same hepatic microsomes irradiated to the total light fluence of 1, 2, 5, 10, 15, 20, and 25 J/cm’, respectively. The P450 content was determined as described under Materials and Methods and found to he 0.89 nmol/mg protein in the unirradiated control sample. This experiment was repeated twice, and spectra from a typical experiment are shown here.

doses of light fluence (-675 nm) in the range of l-25 J/cm2 resulted in significant destruction of P450. The destruction was evident by the reduction in peak height between 450 and 490 nm, and was found to be light dose dependent (Fig. 1). However, these microsomes in the ab- sence of -675 nm light showed a P450 spectrum and P450 content comparable to that obtained with hepatic microsomes prepared from control mice (data not shown). The data shown in Fig. 2 indicate that when hepatic mi- crosomes were irradiated to -675 nm irradiation, AHH, ECD, and ERD activities were also diminished signifi- cantly in a light-dose-dependent manner. The destruction of AHH, ECD, and ERD activities was virtually complete at the dose of 25 J/cm’, the maximum light dose used in this experiment; since enzyme activities were almost un- detectable at this point (Fig. 2).

In order to assess the involvement of ROS in the pho- todestruction of P450 and associated monooxygenase ac- tivities, different concentrations of known quenchers of singlet oxygen (sodium azide, histidine, and 2,5-DMF), superoxide anion (SOD), hydrogen peroxide (catalase), and hydroxyl radical (sodium benzoate, mannitol, and ethanol) were added to these microsomal suspensions prior to their irradiation. For these experiments the total light fluence of 10 J/cm2 was used which was based on the results shown in Figs. 1 and 2, where at this light 6uence greater than 50% destruction of P450 content and

associated monooxygenase activities was observed. As shown in Fig. 3, only the quenchers of singlet oxygen re- sulted in significant protection against the photodestruc- tion of P450 content, and associated AHH, ECD, and ERD activities in these microsomes. While the addition of sodium azide, histidine, and 2,5-DMF prior to irradia- tion showed highly significant protection against photo- destruction in a dose-dependent manner, the addition of SOD, catalase, sodium benzoate, mannitol, and ethanol did not show any protective effect (Fig. 3).

Since lipid peroxidation is thought to be an essential marker for microsomal membrane damage, a major cause of cell death (46,47), in the present study we also deter- mined the effect of irradiating hepatic microsomes ob- tained from AlPcTS-pretreated SENCAR mice on pho- toenhancement of lipid peroxidation. As shown in Fig. 4, irradiation of hepatic microsomes to the =675 nm light caused rapid enhancement of lipid peroxidation in a light- dose-dependent manner, and at 25 J/cm2, a 650% increase over the unirradiated control was observed (Fig. 4). This increase in lipid peroxidation required the presence of light, since these hepatic microsomes in the absence of light showed the same level of lipid peroxidation as that observed in hepatic microsomes prepared from control SENCAR mice (data not shown). To assess the role of ROS in AlPcTS-mediated photoenhancement of lipid peroxidation, hepatic microsomes were irradiated in the presence of various quenchers of ROS. As shown in Fig. 5, only the quenchers of singlet oxygen, i.e., sodium azide, histidine, and 2,5-DMF, resulted in significant protection in a dose-dependent manner. The addition of quenchers

+ AHH

\ \

” - 5 lb 1 0 15 20 25

Light Fluence (J/cm*)

FIG. 2. Light-fluence-dependent in vivo photodestruction of P450- dependent monooxygenase activities in hepatic microsomes obtained from AlPcTS-pretreated SENCAR mice. Microsomes were suspended in 0.1 M phosphate buffer, pH 7.4, and irradiated to various doses of -675 nm light (1 to 25 J/cm’). AHH, ECD, and ERD activities were determined as described under Materials and Methods and found to be 695 + 72 pmol 3-OH-BP/min/mg protein, 334 ? 40 pmol 7-OH-cou- marin/min/mg protein, and 49 + 5 pmol resorufin/min/mg protein in the unirradiated control samples. Data shown are averages of three sep- arate experiments where each assay was conducted in triplicate.

SINGLET OXYGEN-MEDIATED PHOTODYNAMIC EFFECTS 33

ECD

8

-f Histidine - 2,5 DMF -O-SOD + Catalase -h- Sodium benzoate -n- Mannitol --m- Ethanol

Concentration of Quenchers

FIG. 3. Effect of quenchers of ROS on in vivo photodestruction of P450 and associated monooxygenase activities in hepatic microsomes obtained from AlPcTS-pretreated SENCAR mice. Microsomes were suspended in 0.1 M phosphate buffer, pH 7.4, and added with various concentrations of quenchers of ROS prior to their irradiation to a675 nm light (10 J/cm*). The test concentrations of sodium azide, histidine, 2,5-DMF, sodium benzoate, mannitol, and ethanol were in micromolarities, whereas those of SOD and catalase were in pg/ml. The P450 content and AHH, ECD, and ERD activities were determined as described under Materials and Methods and found to be 0.26 k 0.02 nmol/mg protein, 73 f 8 pmol 3- OH-BP/min/mg protein, 138 + 15 pmol ‘I-OH-coumarin/min/mg protein, and 27 + 3 pmol resorufin/min/mg protein, respectively, in these hepatic microsomal preparations irradiated to 10 J/cm* light fluence in the absence of quenchers of ROS. Data from a typical experiment repeated twice with a similar trend are shown here.

of superoxide anion, hydrogen peroxide, and hydroxyl radical showed no protective effect at any concentration used in this study (Fig. 5), suggesting that these species of reactive oxygen are not involved in this process.

In Vitro Studies

In order to further define the capacity of AlPcTS and ~675 nm light to destruct P450, we evaluated P450 con- tent in hepatic microsomes obtained from PB-pretreated adult rats, to which AlPcTS was added followed by ir- radiation. The advantage of using PB-induced hepatic microsomes in the present study was that the P4502Bl isozyme, induced by PB, can easily be probed by the Western blot technique using highly specific monoclonal antibody 2-66-3 directed against the purified enzyme. As shown in Fig. 6, when these hepatic microsomes added with AlPcTS were irradiated to ~675 nm light, signifkant destruction of the P4502Bl isozyme was observed. The destruction was dependent on the concentration of AlPcTS (Fig. 6A) as well as on the total dose of light (Fig. 6B). Furthermore, as shown in Fig. 6C, when these hepatic

microsomes containing AlPcTS were irradiated after the addition of various quenchers of ROS, only the quenchers of singlet oxygen, i.e., sodium azide, histidine, and 2,5- DMF, showed significant protection of P4502Bl isozyme destruction. Similar to the in uiuo studies, the protective effect was dose dependent; however, in a typical Western blot shown in Fig. 6C, only the protection at highest dose level is shown. When these Western blots were subjected to densitometric analysis using a Shimadzu CS-930 TLC scanner with a DR-2 data recorder, scanning data similar to those apparent otherwise were obtained. The addition of 2 pg AlPcTS followed by irradiation (10 J/cm2) resulted in almost 50% destruction of the P4502Bl isozyme; whereas at the highest AlPcTS concentration (12 pg/ml) followed by irradiation (10 J/cm2), or at 2 pg/ml AlPcTS concentration followed by irradiation to a maximum light dose of 30 J/cm2, more than 90% destruction of the P4502Bl isozyme was observed.

Corroborative to in vivo studies, when hepatic micro- somes obtained from control animals were added with AlPcTS followed by ~675 nm irradiation, significant de-

34 AGARWAL ET AL.

800

600

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6 400

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s 200

0

, lb

l-

0 i To 1'5 io i5

Light Fluence (J/cm2)

FIG. 4. Light-fluence-dependent in vivo photoenhancement of lipid peroxidation in hepatic microsomes obtained from AlPcTS-pretreated SENCAR mice. Microsomes were suspended in 0.1 M phosphate buffer, pH 7.4, containing 100 pM MgClr and irradiated to various doses of ~675 nm light (1 to 25 J/cm’). Lipid peroxidation was estimated as described under Materials and Methods and found to be 10.6 + 0.9 nmol MDA/10 min/mg protein in the unirradiated control samples. Data shown are averages of three separate experiments, each conducted in triplicate.

struction of P450 content and associated monooxygenase activities was observed. As shown in Fig. IA, the addition of AlPcTS to these microsomes followed by irradiation (10 J/cm2) resulted in a dose-dependent destruction of P450 content, and AHH, ECD, and ERD activities. Sim- ilarly, when these microsomes were added with 2 pg/ml

80

0 10 20 30 40 50

Concentration of Quenchers

FIG. 5. Effect of quenchers of ROS on in vivo photoenhancement of lipid peroxidation in hepatic microsomes obtained from AlPcTS-pre- treated SENCAR mice. Microsomes were suspended in 0.1 M phosphate buffer, pH 7.4, containing 100 pM MgClz and added with various con- centrations of quenchers of ROS prior to their irradiation to -675 nm light (10 J/cm’). The test concentrations of sodium azide, histidine, 2,5- DMF, sodium benzoate, mannitol, and ethanol were in micromolarities, whereas that of SOD and catalase were in ag/ml. Lipid peroxidation was estimated as described under Materials and Methods and found to be 40.8 f 3.7 nmol MDA/10 min/mg protein in the hepatic microsomal preparation irradiated to 10 J/cm2 light fluence in the absence of quenchers of ROS. Data from a typical experiment repeated twice with similar trend are shown here.

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FIG. 0. AlPcTS-mediated in vitro photodestruction of P4502Bl in hepatic microsomes obtained from PB-pretreated adult rat liver. The left side of the figure represents the Western blot analysis of various samples using monoclonal antibody 2-66-3 directed against the PB-in- duced purified rat liver P450 isozyme. The right side of the figure rep- resents the densitometric analysis of corresponding Western blots using a Shimadzu CS-930 TLC scanner with a DR-2 data recorder. (A) Various concentrations of AlPcTS were added to microsomal suspensions and then the samples were irradiated to a675 nm light (10 J/cm*). Lanes l-3, non-AlPcTS-added unirradiated control sample, AlPcTS-added (2 ag/ml) unirradiated sample, and non-AlPcTS-added irradiated (10 J/ cm’) sample, respectively; lanes 4-12, microsomal suspensions added with 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 pg AlPcTS/ml, re- spectively, and then irradiated to a675 nm light (10 J/cm’). (B) The microsomal suspensions containing AlPcTS (2 pg/ml) were irradiated to various doses of ~675 nm light (l-30 J/cm’). Lanes l-3, same as in A; lanes 4-12, microsomal suspensions added with AlPcTS (2 pg/ml) were irradiated to =675 nm light for the total doses of 1,2,3,5, 10,15, 20, 25, and 30 J/cm’, respectively. (C) The microsomal suspensions containing AlPcTS (2 ag/ml) were added with various quenchers of ROS and then irradiated to -675 light (10 J/cm*). Lanes l-3, same as in A, lane 4, microsomal suspension containing AlPcTS (2 pg/ml) fol- lowed by irradiation (10 J/cm*); lanes 5-12, same as in lane 4 but added with sodium azide, histidine, 2,5-DMF, SOD, cat&se, sodium benzoate, mannitol, and ethanol prior to their irradiation to -675 nm light, re- spectively. The concentration of various quenchers used was 50 pM,

whereas that of SOD and catalase was 50 pg/ml. The amount of protein used in each lane in all the gels was 25 pg. For other details see Materials and Methods. Data from a typical experiment repeated twice with a similar trend are shown here.

concentration of AlPcTS followed by irradiation to light fluence in the range of 1-25 J/cm’, a light-dose-dependent destruction of P450 content and associated AHH, ECD, and ERD activities was apparent (Fig. 7B).

In additional studies, photoenhancement of lipid per- oxidation was also determined in these microsomes by in vitro addition of AlPcTS followed by a675 nm irradiation. As shown in Figs. 8A and 8B, consistent with in vivo results, the addition of AlPcTS to these microsomes fol- lowed by irradiation resulted in significant enhancement of lipid peroxidation. The effect was dependent on both the concentration of AlPcTS (Fig. 8A) and the light flu- ence (Fig. 8B).

Similar to the in viuo studies, only the quenchers of singlet oxygen afforded significant protection against AlPcTS-mediated photodestruction of P450 content, and AHH, ECD, and ERD activities, as well as photoen-

SINGLET OXYGEN-MEDIATED PHOTODYNAMIC EFFECTS 35

FIG. 7. AlPcTS-mediated in vitro photodestruction of P450 content, and associated monooxygenase activities in lepatic microsomes obtained from control SENCAR mice. (A) Various concentrations of AlPcTS were added to the microsomal suspension in 0.1 M phosphate buffer, pH 7.4, and then samples were irradiated to ~675 nm light (10 J/cm’). (B) The microsomal suspensions in 0.1 M phosphate buffer, pH 7.4, containing AlPcTS (2 ag/ml) were irradiated to various doses of ~675 nm light (1 to 25 J/cm’). The P450 content, and AHH, ECD, and ERD activities were determined as described under Materials and Methods and found to be, respectively, 0.85 + 0.06 nmol/mg protein, 660 + 63 pmol3-OH- BP/min/mg protein, 346 + 47 pmol 7-OH-coumarin/min/mg protein, and 43 + 7 pmol resorufin/min/mg protein in the non-AlPcTS-added unirradiated control samples. Data shown are averages of three separate experiments where each assay was conducted in triplicate.

A d P-450 -4-a AHH _.* .I*-.. EQ-J --A--- ERD

0-l I 0 2 4 6 8 10

B + P-450 -a-* AHH

0 5 10 15 20 25

AlPcTS @g/ml) Light Fluence (Jkm2)

hancement of lipid peroxidation in these microsomal sus- pensions, whereas the quenchers of superoxide anion, hy- drogen peroxide, and hydroxyl radical did not show any change (data not shown).

DISCUSSION

Several lines of evidence have shown that the admin- istration of phthalocyanines to animals results in their localization in membranous structures like plasma mem- branes, and subcellular organelle membranes such as nu- clei, mitochondria, lysosomes, and microsomes (26, 48, and references therein). It is therefore widely accepted that membranes are principal targets of phototoxic effects associated with phthalocyanine-mediated photodynamic processes (26 and references therein). We have recently shown that in vitro incubation of epidermal microsomes with AlPcTS followed by irradiation with a suitable wavelength of light causes disruption of endoplasmic re- ticulum membranes (18, 19). The data obtained in the present study extend the previously published observa- tions and suggest that the parenteral administration of AlPcTS to animals leads to its localization in the hepatic endoplasmic reticulum, and thereby these membranes become highly photosensitive. The exposure of these membrane preparations to ~675 nm light, therefore, re- sults in the extensive damage of both the membrane- bound proteins and the lipid domains. Such photodamage was apparent by a significant decrease in (a) membrane- bound hemeprotein P450, (b) P450-dependent monoox- ygenase activities such as AHH, ECD, and ERD, and (c) P4502Bl reactivity in the Western blot analysis. Addi- tionally, this photodamage resulted in a several-fold in- crease in lipid peroxidation as measured by the formation

of thiobarbituric acid reacting species. The results of the present study further demonstrate that less than 1 pg concentration of AlPcTS/mg hepatic microsomal protein, achieved within 24 h after 5 mg/kg ip administration of AlPcTS (49), causes comparable damage as observed by the exogenous addition of 4 pg AlPcTS/mg hepatic mi- crosomal protein, suggesting that the pharmacodynamics of AlPcTS play an important role in its effective local- ization in the membrane.

The photosensitizer and light-mediated damage of en- doplasmic reticulum membranes observed in the present study may particularly be important in the cascade of events during the PDT, since it results in the inability of microsomes to sequester effectively the [Ca2+]i from cy- tosol, thus leading to an increase in cytosolic [Ca2+li (50). This is further supported by a recent observation by Ben- Hur et al. (51) showing a rapid transient increase in cy- tosolic [Ca’+]i from about 0.2 PM to 1.0 mM after the ir- radiation of Chinese hamster cells preloaded with chlo- roaluminum phthalocyanine. The increase in cytosolic [Ca2+]i has widely been accepted to play an important role in the manifestation of toxicity of various chemicals, ultimately leading to cell death (52).

The initial step following exposure of the photosensi- tizers, including phthalocyanines, to the light is the ab- sorption of energy and subsequent conversion of photo- sensitizer to an electronically excited state which is usually a singlet state (4, 26). Since a photosensitizer in the singlet state is an extremely short-lived species (half- life , ~10~~ to lo-’ s), it emits radiation to enter a more stable and longer lived triplet excited state (half-life, = lop3 s) (4 and references therein). The phthalocyanine- mediated reactions of biological importance are typically

36 AGARWAL ET AL.

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200- r’C--d’

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AlPcTS @g/ml) Light Fluence (J/cm*)

FIG. 8. AlPcTS-mediated in vitro photoenhancement of lipid peroxidation in hepatic microsomes obtained from control SENCAR mice. (A) Various concentrations of AlPcTS were added to the microsomal suspension in 0.1 M phosphate buffer, pH 7.4, containing 100 pM MgCI, and then samples were irradiated to a675 nm light (10 J/cm2). (B) The microsomal suspensions in the same buffer containing AlPcTS (2 pg/ml) were irradiated to various doses of -675 nm light (1 to 25 J/cm2). Lipid peroxidation was estimated as described under Materials and Methods and found to be 10.9 f 1.1 nmol MDA/10 min/mg protein in the non-AlPcTS-added unirradiated control samples. Data shown are averages of three separate experiments, each conducted in triplicate.

carried out by the intermolecular interaction of the pho- tosensitizer in the triplet state with molecular oxygen in the ground state, leading to the generation of ROS (4 and references therein). The triplet energy of phthalocyanines (26-30 kcal/mol) appears to be sufficient for the gener- ation of singlet oxygen (23 kcal/moi) through the inter- molecular energy transfer involving the reactions termed Type II reactions (53). The exact sequences involved in these reactions in reference to AlPcTS are outlined below.

AlPcTS (ground state) + hu + lAlPcTS (singlet)

‘AlPcTS + 3AlPcTS (triplet)

3A1PcTS + 302 (triplet) + AlPcTS + ‘02 (singlet)

However, under the Type I reaction, the possibility of transfer of an electron or a hydrogen atom from phthal- ocyanine in the triplet state to molecular oxygen or other molecules lying in the close vicinity through the redox reactions, leading to the generation of superoxide anion, hydroxyl radical, and/or peroxides, cannot be ruled out (53).

The absolute requirement of molecular oxygen during the phthalocyanine-mediated PDT is well established since cellular damage is minimal or absent under hypoxic/ anoxic conditions (28). The ROS generated via Type I and/or Type II reactions may therefore play an important role in the photocatalytic disruption of malignant tissue undergoing phthalocyanine-mediated PDT. Nevertheless no unifying concept so far has emerged to suggest the definite role of any particular ROS in this process. How- ever, in the present study the effectiveness of only sodium azide, histidine, and 2,5-DMF (the known quenchers of singlet oxygen) to protect against both the AlPcTS-me- diated photodestruction of P450 and associated mono-

oxygenase activities, and photoenhancement of lipid per- oxidation strongly suggests the involvement of singlet oxygen in this process. Since in our study the scavengers of superoxide anion, hydrogen peroxide, and hydroxyl radical did not afford any protection, the involvement of any other ROS in this process can be ruled out. In sum- mary our data suggest that AlPcTS-mediated photo- damage leads to the destruction of both membrane protein and lipid components, and that singlet oxygen is involved in this process.

REFERENCES 1. Dougherty, T. J. (1984) CRC Crit. Reu. 2,83-116. 2. Dougherty, T. J., Potter, W. R., and Bellnier, D. (1990) in Photo-

dynamic Therapy of Neoplastic Diseases (Kessel, D., Ed.), Vol. 1, pp. l-19, CRC Press, Boca Raton, FL.

3. Dougherty, T. J. (1989) Sem. Surg. Oncol. 5,6-16. 4. Gomer, C. J., Rucker, N., Ferrario, A., and Wong, S. (1989) Radiat.

Res. 120, l-8.

5. Dougherty, T. J. (1987) Photo&em. Photobid. 46,569-574. 6. Kessel, D. (1986) Photo&em. Photobid. 44,193-196. 7. Moan, J., Waksvik, H., and Christensen, T. (1980) Cancer Res. 40,

2915-2918.

8. Atlante, A., Morano, G., Passarella, S., and Salet, C. (1986) &&em. Biophys. Res. Commun. 141,X&590.

9. Sandberg, S., and Romslo, P. (1981) Clin. Chim. Acta 109, 193- 201.

10. Das, M., Dixit, R., Mukhtar, H., and Bickers, D. R. (1985) Cancer Res. 45,608-615.

11. Dixit, R., Mukhtar, H., and Bickers, D. R. (1983) Photo&em. Pho- tobid. 37,173-176.

12. Das, M., Mukhtar, H., Greenspan, E. R., and Bickers, D. R. (1985) Cancer Res. 46,6328-6330.

13. Dixit, R., Mukhtar, H., and Bickers, D. R. (1983) J. Iwest. Dermatd. 81,369-375.

14. Athar, M., Mukhtar, H., and Bickers, D. R. (1988) J. Znuest. Der- matol. 90, 662-657.

SINGLET OXYGEN-MEDIATED PHOTODYNAMIC EFFECTS 37

15.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32.

Gomer, C. J., and Razum, N. J. (1984) P~otochem. Photobid. 40, 435-439.

Athar, M., Agarwai, R., Bickers, D. R., and Mukhtar, H. (1991) in Pharmacology of the Skin (Mukhtar, H., Ed.), pp. 269-279, CRC Press, Boca Raton, FL.

Mitchell, J. B., McPhearson, S., DeGraff, W., Gamson, J., Zabell, A., and Russo, A. (1985) Cancer Res. 46,2009-2011.

Agarwal, R., Athar, M., Bickers, D. R., and Mukhtar, H. (1990) Biochem. Biophys. Res. Commun. 173, 34-41. Agarwal, R., Athar, M., Urban, S. A., Bickers, D. R., and Mukhtar, H. (1991) Cancer L&t. 56,125-129.

Bodaness, R. S., and Chan, P. C. (1977) J. Biol. Chem. 262,8554- 8560. Buettner, G. R., and Oberley, L. W. (1980) FEES I&t. 121, 161- 164.

Weishaupt, K., Gomer, C. J., and Dougherty, T. (1976) Cancer Res. 36,2326-2329. Keene, J. P., Kessel, D., Land, E. J., Redmond, R. W., and Truscott, T. G. (1986) Photo&em. Photobid. 43, 117-120.

Buettner, G. R., and Need, M. J. (1985) Cancer I.&t. 25, 297-304.

van Lier, J. E. (1990) in Photodynamic Therapy of Neoplaatic Dis- eases (Kessel, D., Ed.), Vol. 1, pp. 279-290, CRC Press, Boca Raton, FL.

Rosenthal, I. (1991) Photo&em. Photobiol. 53,859-870.

Ben-Hur, E., and Rosenthal, I. (1985) Znt. J. R&at. Bid. 47,145- 147.

Brasseur, N., Ali, H., Autenrieth, D., Langlois, R., and van Lier, J. (1985) Photo&m. Photobiol. 42,515-521.

Chan, W. S., Svensen, R., Philips, D., and Hart, I. R. (1986) Br. J. Cancer53,255-263.

Chan, W. S., Marshall, J. F., and Hart, I. R. (1987) Photo&m. Photobid. 46,867-872.

Chan, W. S., Marshall, J. F., Lam, G. Y. F., and Hart, I. R. (1988) Cancer Res. 48,3040-3044.

Tralau, C. J., Barr, H., Sandeman, D., Barton, T., Lewin, M., and Bown, S. (1987) Photo&em. Photobiol. 46, 777-782.

33.

34.

35. 36. 37. 38.

39.

40.

41.

42.

43.

44. 45.

46.

47. 48.

49.

50.

51.

52.

53.

Wang, Z. Y., Das, M., Bickers, D. R., and Mukhtar, H. (1988) Drug Metab. Dispos. 16,98-103. Agarwal, R., Wang, Z. Y., Bik, D. P., and Mukhtar, H. (1991) Drug Metab. Dispos. 19,620-624. Bradford, M. D. (1976) And. Biochem. 72,248-256. Omura, T., and Sato, R. (1964) J. Bid. Chem. 239, 2370-2378.

Laemmli, U. K. (1970) Nature 227,68&685. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. N&l. Acad. Sci. USA 76,4350-4354.

Mukhtar, H., AgarwaI, R., and Bickers, D. R. (1991) in Pharmacology of the Skin (Mukhtar, H., Ed.), pp. 89-109, CRC Press, Boca Raton, FL.

Khan, W. A., Park, S. S., Gelboin, H. V., Bickers, D. R., and Mukhtar, H. (1989) J. Pharmacol. Erp. Ther. 249,921-927.

Nebert, D. W., and Gelboin, H. V. (1968) J. Biol. Chem. 243,6242- 6249.

Greenlee, W. F., and Poland, A. (1978) J. Phurmacol. Ezp. Ther. 206,596-605. Bickers, D. R., Choudhury, T. D., and Mukhtar, H. (1982) Mol. Pharmacol. 2 1,239-247. Pohl, R. J., and Fouts, J. R. (1980) Anal. Biochem. 107,150-155. Wright, J. R., Colby, H. D., and Miles, P. R. (1981) Arch. Biochem. Biophys. 206, 296-304.

Bus, J. S., and Gibson, J. R. (1979) Rev. Biochem. Toxicol. 1, 125- 149. Girotti, A. W. (1990) Photochem. Photobid. 51,497-509. Robinson, R. S., Roberts, A. J., and Campbell, I. D. (1987) Photo- them. Photobiol. 45, 231-234.

Agarwal, R., Athar, M., Elmets, C. A., Bickers, D. R., and Mukhtar, H. (1992) Photo&em. Photobiol., in press.

Thor, H., Hartzell, P., Svensson, S. A., Orrenius, S., Mirabelli, F., Mnarinoni, V., and Bellomo, G. (1985) Biochem. Phurmacol. 34, 3717-3723. Ben-Hur, E., Dubbehnan, T. M. A. R., and Van Steveninck, J. (1991) Photo&em. Photobiol. 54, 163-166.

Di Monte, D., Bellomo, G., Thor, H., Nicotera, P., and Orrenius, S. (1984) Arch. B&hem. Biophys. 236, 343-350. Spikes, J. D. (1986) Photo&m. PhotobioZ. 43, 691-699.