PHOTODYNAMIC THERAPY WITH PHOTOFRIN II INDUCES PROGRAMMED CELL DEATH IN CARCINOMA CELL LINES

Abstract The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD₅₀). In contrast, PC3 showed only marginal response at the LD₅₀ but had a marked response at the LD₈₅. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy-induced cell death, this response is not universal for all cancer cell lines.     

Initiation of Apoptosis versus Necrosis by Photodynamic Therapy with Chloroaluminum Phthalocyanine

Abstract— While chloroaluminum phthalocyanine is a highly effective photosensitizer of murine leukemia P388 or L1210 cells, the mode of cell death varies as a function of the PDT dose. When cells were incubated with 0.3 mUM of the sensitizer, a light dose of 45 mJ cm_-2 (670 5 nm) yielded a 90% apoptotic cell population within 60 min. The sensitizer localized throughout the cytoplasm and catalyzed both lysosomal and mitochondrial photodamage at this light dose. Higher light doses yielded progressively more membrane photodamage and inhibited the apoptotic response as determined by the examination of Hochst dye HO 33342-IabeIed nuclei, DNA fragmentation on gels and a poly(adenosylribose) polymerase (PARP)-cleavage assay. Pulse-field gel electrophoresis revealed nonspecific DNA degradation to particles 50 kbp at the higher PDT doses but neither PARP cleavage nor apoptotic nuclei     

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

Abstract— Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitiz-er Pc 4 (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome-size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotox-icity.     

DOES MEMBRANE ATTACKING PHOTODYNAMIC ACTION REFLECT THE SO-CALLED PHASE TRANSITION?

Abstract— With Saccharomyces cerevisiae cells the temperature dependence of toluidine blue (TB) sensitized photodynamic inactivation was measured in the range 10–33°C using either H₂O (pH 7.9) or 45% D₂O (pH 6.8) as a solvent. Plots of ln(1/LD₅₀) (LD₅₀: light dose for 50% survival) as a function of the reciprocal of temperature could be fitted by two straight lines with a discontinuity at 21–22°C. In view of previous findings on TB-cell interaction that TB sensitizes the cell, after long incubation, in the membrane rather than from outside the cell, it is suggested that the present results reflect the phase transition of lipid matrix.     

In situ COMPARISON OF 665 nm and 633 nm WAVELENGTH LIGHT PENETRATION IN THE HUMAN PROSTATE GLAND

Abstract— The depth of treatment in photodynamic therapy (PDT) of tumors varies with the wavelength of light activating the photosensitizer. New generation photosensitizers that are excited at longer wavelengths have the potential for increasing treatment depths. Tin ethyl etiopurpurin (SnET2), a promising second-generation photosensitizer is maximally activated at 665 nm, which may be significantly more penetrating than 633 nm light currently used with porphyrins in PDT. The penetration of 665 nm and 633 nm wavelength red light in the prostate gland was compared in 11 patients undergoing prostatic biopsies for suspected prostatic cancer. Interstitial optical fibers determined the light attenuation within the prostate gland. Of the 11 patients, 7 had dual wavelength and 4 had single wavelength studies. The mean attenuation coefficients, μ_eff, for 665 nm and 633 nm wavelength light were 0.32 ± 0.05 mm^-1 and 0.39 ± 0.05 mm^-1, respectively, showing a statistically significant difference (P = 0.0003). This represented a 22% increase in the mean penetration depth and at 10 mm from the delivery fiber there was 1.8 times as much 665 nm light fluence than 633 nm. The mean μ_eff at 665 nm for benign and malignant prostate tissue were similar ( P = 0.42), however, there was significant interpatient variation (μ_eff ranging from 0.24 to 0.42 mm^-1) reflecting biological differences of therapeutic importance. The enhanced light fluence and penetration depth with 665 nm light should allow significantly larger volumes of prostatic tissue to be treated with SnET2-mediated PDT.     

SITES OF PHOTODYNAMICALLY INDUCED DNA REPAIR IN HUMAN CELLS

Abstract Human REH cells were incubated with the photosensitizers meso -tetra(4-sulfonatophenyl)porphyrin (TSPP=TPPS₄) or meso -tetra(3-hydroxyphenyl)porphyrin (3-THPP). The relatively hydrophilic TSPP was partly found in the cytoplasm and partly in the nuclei, whereas the lipophilic 3-THPP was found apparently in membranes and not inside the nuclei. After illumination, sites of DNA repair were labeled by means of a monoclonal antibody against proliferating cell nuclear antigen (PCNA) bound in the nuclei. The amount of bound PCNA in non-S-phase cells was proportional to the light dose. The bound PCNA was homogeneously distributed in the nuclei 0.5 h after photodynamic treatment (PDT) with TSPP. In contrast, for cells given PDT with 3-THPP, the periphery of the nuclei was selectively labeled, indicating that the initial DNA damage was localized close to the sensitizer at the nuclear membrane.     

THE INFLUENCE OF SODIUM PENTOBARBITAL ANESTHESIA ON in vivo PHOTODYNAMIC THERAPY

Abstract The use of sodium pentobarbital anesthesia 50 jig gm⁻¹ during localized photodynamic therapy (PDT) was examined in C57BL/6 mice transplanted with the pigmented B-16 melanoma. A 10 mg kg⁻¹ i.p. injection of Photofrin II was administered 24 h prior to light exposure (630 nm, 150 mW, cm⁻², 300-500 J cm⁻²). Separate groups of mice were utilized to monitor tumour temperature and PDT tumor response. Core tumor temperatures decreased by approx. 10^oC following sodium pentobarbital administration. Tumor responses were determined by documenting the percentage of treated animals without tumor recurrences for a period of 50 days following PDT. Superior PDT induced tumor responses were obtained in control (non-anesthetized) mice following light doses of 400 and 500 J cm⁻². The results of this study indicate that sodium pentobarbital can induce a protective effect on B-16 melanomas treated with PDT.     

PHOTODYNAMIC INACTIVATION OF E. COLI BY ROSE BENGAL IMMOBILIZED ON POLYSTYRENE BEADS

Abstract. The photodynamic inactivation of E. coli by visible light and O₂ was found to occur in the presence of the sensitizer rose bengal, immobilized by covalent bonding to polystyrene beads. The demonstrated absence of significant amounts of dissolved rose bengal indicated that an inactivation mechanism based on penetration of sensitizer molecules into the cell's interior could not be operating. Survival curves typically exhibited induction periods followed by rapid exponential death, with 99.99% kill requiring 1–2 h depending on conditions. A mechanism involving the participation of photo-generated singlet excited oxygen O₂(¹δ) in inactivation of E. coli is proposed. The photodynamic inactivation rate increased significantly in H₂O compared with H₂O, which is evidence supporting singlet oxygen as an active intermediate, since O₂(¹δ) has a much longer lifetime in H₂O than in H₂O. H₂O did not act as a short term poison in the absence of sensitizer.     

Protein-A-mediated Targeting of Bacteriochlorophyll-IgG to Staphylococcus aureus: A Model for Enhanced Site-Specific Photocytotoxicity

Abstract— A model for studying the efficiency of photodynamic action with a photosensitizer placed exclusively on the bacterial cell wall has been used. Bacteriochlorophyllide molecules, conjugated to rabbit immunoglobulin G (IgG), were synthesized. The conjugated pigment bacteriochlo-rophyll (Bchl)-IgG bound with high specificity to protein-A residues naturally exposed on the cell wall of the bacterium Staphylococcus aureus Cowan I. In bacterial suspensions the phototoxicity of the targeted conjugates (0.5-2.5 pigment per IgG molecule) was dose dependent (LD₅₀= 1.7 μ M ) in the presence of light (Λ > 550 nm) and inhibited by native IgG but not by ovalbumin, suggesting selective interaction with protein-A on the bacterial cell wall. No dark toxicity was noticed even with the highest conjugate concentration tested. In contrast, the photocytotoxicity of bacteriochlorophyll-serine (Bchl-Ser, LD₅₀= 0.07 μ M ) used as a nontargeted control was not inhibited by IgG. In spite of its lower apparent potency, Bchl-IgG was found to be 30 times more efficacious than Bchl-Ser: At LD₅₀, only 66000 Bchl-IgG molecules were bound per bacterium compared to 1900 000 molecules of Bchl-Ser. The higher efficacy of Bchl-IgG is explained by its exclusive position on the bacterial cell wall. Consequently, photogeneration of oxidative species is confined to the cell wall and its vicinity, a seemingly highly susceptible domain for photodynamic action. In considering the design of cell-specific sensitizers for bacterial and cancer therapies, it would be beneficial to identify the more discretely sensitive subcellular domains as targets.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

A TEST OF DIFFERENT PHOTOSENSITIZERS FOR PHOTODYNAMIC TREATMENT OF CANCER IN A MURINE TUMOR MODEL

Abstract The efficiency of different sensitizers for photodynamic therapy (PDT) was tested using a model system with a C3H mammary carcinoma growing subcutaneously on the dorsal side of mouse feet. Growth curves were constructed from which growth delay and doubling time in the regrowth phase were calculated. As PDT induced oedema in the mouse foot, this model system also allowed assessment of normal tissue response.
The following sensitizers were tested: hematoporphyrin derivative (HpD), Photofrin II (PII), tetraphenylporphinetetrasulfonate (TPPS₄), acridine orange (AO), phthalocyanine tetrasulfonate (PCTS), Al- and Zn-phthalocyanine tetrasulfonate (A1PCTS and ZnPCTS). For tumor control, the following sensitizer efficiencies were found: PII > HpD > AIPCTS > TPPS₄ >>> ZnPCTS, PCTS, AO. With regard to sensitizing normal-tissue damage: PII > AIPCTS, TPPS₄ > HpD, ZnPCTS, PCTS. The results suggest that AIPCTS should be further evaluated for use in PDT.     

Photodynamic Therapy with a Diode Laser for Implanted Fibrosarcoma in Mice Employing Mono-L-Aspartyl Chlorin E6

Abstract— The authors performed photodynamic therapy (PDT), avoiding any hyperthermic effects, using a newly developed diode laser and photosensitizer, mono-L-aspar-tyl chlorin e₆ (NPe6), of Meth-A fibrosarcoma implanted in mice and achieved tumor therapeutic benefit. The photodynamic light treatment was performed 5 h following the photosensitizer administration. With 5.0 mg/kg NPe6 and light doses of 50, 100, 150 and 200 J/cm², the tumor cure rates were 20, 50, 70 and 90%, respectively. With 100 J/cm² laser exposure and NPe6 doses of 1.25, 2.5, 5.0, 7.5 and 10.0 mg/kg, the tumor cure rates were 0, 20, 50, 70 and 90%, respectively. A charge-coupled device (CCD) camera system was employed to measure the NPe6 fluorescence intensity correlating with the residual amount of the photosensitizer at deferent depth from the tumor surface. The ratios of the NPe6 fluorescence intensity at 3 mm from the tumor surface following 50, 100, 150 and 200 J/cm² laser exposure to no laser exposure were 0.73, 0.36, 0.22 and 0.16, respectively. With samples sectioned at 1 mm depth, after 50 J/cm² and the same photosensitizer dose (5 mg/kg) this ratio was 0.19. These results suggest that a certain increase in the tumor tissue level of NPe6 and a certain increase of laser light dose reaching deeper layers of tumor caused an increase in percent cure. In addition, the effectiveness of PDT depends on the total laser dose reaching deeper layers of tumors. Furthermore, the effectiveness of PDT tends to correlate with the amount of NPe6 photobleaching by PDT.     

PRIMARY DNA DAMAGE, HPRT MUTATION AND CELL INACTIVATION PHOTOINDUCED WITH VARIOUS SENSITIZERS IN V79 CELLS

DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3—hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso -tetra(4—sulfonatophenyl)porphine (TPPS₄) or the cationic dye meso -tetra( N -methyl-4-pyridyl)porphine ( p -TMPyPH₂ before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS₄ was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS₄ and p -TMPyPH₂ was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS₄ was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p -TMPyPH₂. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

PHOTOBLEACHING OF MONO-l-ASPARTYL CHLORIN e6 (NPe6): A CANDIDATE SENSITIZER FOR THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract— Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono- l -aspartyl chlorin e₆ (NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 × 10⁻⁴; this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 × 10 ⁵ and 2.8 × 10⁻⁵, respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 × 10 ⁻⁶), while the Zn derivative was more sensitive (yield = 1.9 × 10⁻²). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photoblcaching two- to four-fold, while the electron acceptor, met-ronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.     

Singlet Oxygen Generation by Metallotexaphyrins

Abstract. Metallotexaphyrins have clinical applications as photo-sensitizers of photodynamic therapy (PDT). The singlet oxygen quantum yield (φ>_Δ) was determined for a series of metallotexaphyrin derivatives (Lu [III], Y [III], Cd [II], In [III] and Gd [in]) under conditions where the agents are believed to exist in monomeric form. The results show φ_Δ of metallotexaphyrins vary with the medium and the metal cation. Measurements on the Lu (III) texaphyrin led to φ_Δ= 0.38 in unbuffered 5% Tween 20 and φ_Δ= 0.58 in pH 7.4 phosphate buffer plus 1% Triton X-100 (±10%). The in vitro photodynamic efficiency calculated from φ_Δ is compared to in vivo PDT efficacy in an animal tumor model.     

THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS

Abstract— The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase-locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators.
The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different light doses designed to span the dose-response range of fluorescein fluorescence exclusion (measured 8–10 min after fluorescein injection). The assay validation experiment used an anionic photosensitizer, 2-[l-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 μmol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose-response model to the data were: median fluorescence exclusion light dose FE₅₀= 275 ± 8.3 J/cm² and Hill sigmoidicity parameter m =−3.66 ± 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates. The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.     

DNA Damage and Apoptosis Induced by Photosensitization of 5,10,15,20-Tetrakis (N-methyl-4-pyridyl)-21H,23H-porphyrin via Singlet Oxygen Generation

Cancer photodynamic therapy (PDT) requires photosensitizers that efficiently and selectively destroy tumor cells. We investigated 5,10,15,20-tetrakis ( N -methyl-4-pyridyl)-21 H ,23 H -porphyrin (TMPyP) as a potential cancer treatment. Confocal fluorescence microscopy showed that TMPyP was localized in the nuclei, whereas 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PPIX) was localized diffusely in the cytoplasm of human leukemia (HL-60) cells. In HL-60 cells under UVA irradiation, TMPyP effectively induced apoptosis. Moreover, 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative product of 2'-deoxyguanosine, was accumulated in the DNA of cells treated with photoirradiated TMPyP, whereas only small amounts were observed in ALA-treated cells in the presence of UVA light. TMPyP and UVA caused extensive damage at every guanine residue in DNA fragments obtained from the human p 53 tumor suppressor gene and the c-Ha- ras -1 proto-oncogene, whereas PPIX induced little DNA damage under these conditions. Electron spin resonance spectroscopy using a singlet oxygen (¹O₂) probe and D₂O showed that photoexcited TMPyP generated ¹O₂. These results suggest that photoexcited TMPyP reacts with oxygen to generate ¹O₂, which in turn, oxidizes guanine residues. Taken together, the results demonstrated that TMPyP was localized in the nucleus where it was photosensitized to induce DNA damage, suggesting that TMPyP may have clinical utility as a nucleus-targeted PDT.     

Photodynamic Therapy of Human Glioma (U87) in the Nude Rat

Abstract— We measured the response of normal brain and the human U87 glioma implanted in the brain of rats (n = 65) to photodynamic therapy (PDT) using Photofrin as the sensitizer. Normal brain and U87 tumor implanted within brain of athymic (nude) rats were subjected to PDT (12.5 mg/kg of Photofrin) at increasing optical energy doses (35 J/cm², 140 J/cm², 280 J/cm²) of 632 nm light. Photofrin concentration in tumor, brain adjacent to tumor and normal brain were measured in a separate population of rats. Twenty-four hours after PDT, the brains were removed, sectioned, stained with hematoxylin and eosin (H&E), and the volumes of the PDT-induced lesion measured. Photofrin concentration in tumor greatly exceeded that of normal brain and brain adjacent to tumor (>20×). Both normal brain and U87 tumor exhibited superficial tissue damage with PDT at 35 J/cm². However, both normal and tumor-implanted brain exhibited tissue damage with increasing optical dose. A heterogeneous pattern of pannecrosis along with a uniform volume of pannecrosis was detected in the tumor. In contrast, normal brain exhibited a uniform sharply demarcated volume of necrosis. Our data indicate that the U87 human brain tumor model and the normal brain in the athymic rat are sensitive to PDT and Photofrin with an optical dose-dependent response to treatment.