CATALASE INACTIVATION FOLLOWING PHOTOSENSITIZATION WITH TETRASULFONATEDMETALLOPHTHALOCYANINES

Abstract— Catalase (CAT) in solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during photosensitization with tetrasulfonated metallophthalocyantnes (MePcS₄). The effect of added scavengers and D₂0 showed that both singlet oxygen and free radical species are involved in this process. Evidence was found that direct interactions of ground or excited-stated photosensitizers with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized protein damage included oxidation of cysteine residues as well as other amino acids, as demonstrated by the formation of carbon-centered free radicals and the loss of absorbance at λ= 275 nm. In parallel, we detected degradation of the CAT heme groups, accompanied by release of Fe(II) ions in solution. These combined phenomena initiate cross-linkages between CAT subunits and subsequent degradation of the protein with formation of irreversible aggregates in solution. Phthalocyanine-mediated photoinactivation of cell-bound CAT results in loss of protection against accumulating H₂0₂, providing an additional pathway of phototoxicity.     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN: III. ROLE OF PLASMA PROTEINS IN PHOTOSENSITIZER CLEARANCE FROM CELLS

Abstract— Human plasma proteins, albumin, globulins and low density (LDL), high density (HDL) and very low density (VLDL) lipoproteins were tested for their effects on retention of Photofrin and three other photosensitizers in cultured cells. This was assessed by incubating the cells, subsequent to the exposure to Photofrin, in the photo-sensitizer-free medium containing various concentrations of different plasma proteins. Photofrin clearance levels differed with individual plasma proteins and also were dependent on concentration of these proteins in the incubation medium. All of the proteins except VLDL promoted clearance of Photofrin taken up by the cells in the presence of 5% human serum. Subsequent to some Photofrin exposure conditions (in the presence of 5% fetal bovine serum, or in protein-free medium), albumin, in contrast to LDL, HDL and globulins, exhibited decreased capacity for promoting the photosensitizer clearance from the cells. The VLDL showed very little or no effect in promoting cellular clearance of Photofrin, tetraphenyl porphine tetrasulfonate (TPPS₄), and di- and tetrasulfonated chloroaluminum phthalocy-anine (AlPcS₂ and AlPcS₄, respectively). The LDL seem to be particularly effective in promoting clearance of Photofrin and AlPcS₂ from the cells, whereas albumin and globulins were shown to be more effective than LDL and HDL in promoting the cellular clearance of TPPS₄.     

In vitro PHOTODYNAMIC EFFECTS OF LYSYL CHLORIN p6: CELL SURVIVAL, LOCALIZATION AND ULTRASTRUCTURAL CHANGES

The in vitro cell survival, localization and ultrastructural changes following irradiation were examined in 9L glioma cells sensitized with a new photosensitizer, lysyl chlorin p₆ (LCP). In clonogenic assays, LCP was 10–100-fold more phototoxic than photofrin II on a μg/mL basis. Lysyl chlorin p₆ uptake was blocked when cells were incubated at 2°C. In view of the chemical properties of LCP, this finding indicates that uptake probably occurred through the endocytic pathway. Fluorescence studies showed LCP localized in a region of the endocytic compartment similar in size, shape and distribution to that labeled by lucifer yellow CH (LY), as well as localizing diffusely throughout the perinuclear cytoplasm. Cells stained with both LY and LCP, however, had distinctly separate regions of staining. Lysyl chlorin p₆ localization differed from that of fluorescent probes labeling the mitochondria, Golgi apparatus and endoplasmic reticulum. Ultrastructural changes at both 2 and 30 min after laser irradiation were similar. Mitochondria were often condensed or swollen and also had constrictions and cytoplasmic invaginations. The Golgi apparatus, perinuclear space and rough endoplasmic reticulum (RER) were dilated. These data demonstrate that LCP localizes in a portion of the endosomal compartment, but that morphologic damage initially occurs in the mitochondria, Golgi apparatus and RER.     

Distribution of disulfonated and tetrasulfonated aluminum phthalocyanine between malignant and host cell populations of a murine fibrosarcoma

Levels of disulfonated and tetrasulfonated aluminum phthalocyanines (AlPcS_2,4) were measured in cells derived from FsaR tumors (murine fibrosarcoma using a fluorescence-activated cell sorter (FACS). The tumors were excised from animals injected with the sensitizer 24 h earlier and enzymatically dissociated. Before flow cytometry, the cells were stained with fluorescein isothiocyanate-conjugated anti-mouse monoclonal antibodies to specific immune cell membrane markers (Mac1, Fc receptor (FcR) or CD45). Staining to FcR and CD45 was combined with a DNA stain Hoechst 33342. This enabled concomitant discrimination to be made by the FACS between different populations of tumor-infiltrating host cells and malignant cells. The results showed on average 1.49 times higher AlPcS₂, levels and 1.16 times higher AlPcS₄ levels in Mac1-positive (Mac1⁺) compared with Mac1-negative (Mac1⁻) tumor cell populations. The same type of experiments performed with SCCVII tumor (squamous cell carcinoma) gave average Mac1⁺/Mac1⁻ ratios of 1.75 and 1.45 for AlPcS₂ and AlPcS₄ respectively. The data using other antibodies and DNA staining are consistent with the conclusion that, based on average per cell content, elevated levels of AlPcS₂, and to a lesser extent AlPcS₄, are retained in tumor-associated macrophages (TAM). The levels of these photosensitizers in other leukocytes and in non-immune host cells were not substantially different from those in malignant tumor cells. It is also shown that elevated levels of AlPcS₂ and AlPcS₄ are not localized in all TAM, but rather in a fraction of this cell population characterized by extremely high photosensitizer content.     

PHTHALOCYANINE FLUORESCENCE AT HIGH CONCENTRATION: DIMERS OR REABSORPTION EFFECT?

Abstract— For tetrasulfonated aluminum phthalocyanine (AlPcS₄), dimer formation is characterized in the absorption spectrum by a broadening of the Q-band and the appearance of a new band at the red edge of the spectrum. The high concentrations required to produce dimers, however, often leads to anomalous observations in fluorescence spectroscopy. In the present study, we have examined the photophysical characteristics of two dye systems; AlPcS₄ in a 66% ethanol/water mixture and disulfonated aluminum phthalocyanine in methanol. Using absorption spectroscopy, the formation of dimers is shown to be prevalent only in the case of AlPcS₄. The fluorescence emission spectra in both cases, however, exhibit similar spectral changes with increasing dye concentration. The measured fluorescence decay profiles for both dyes also show similar trends: They are monoexponential, invariant with emission wavelength and have decay times that increase with dye concentration. These distortions are sometimes incorrectly attributed to dimer fluorescence. We find no evidence for the existence of dimer fluorescence and demonstrate that these data can be readily explained, by taking into consideration the effects of reabsorption of fluorescence.     

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.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

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.     

The Effect of Charge on Cellular Uptake and Phototoxicity of Polylysine Chlorine6Conjugates

Abstract— The effect of charge on the cellular uptake, localization and phototoxicity of conjugates between chlorin_e6 ( c _e6) and poly-L-lysine was studied in vitro. These conjugates (average MW35–55 kDa) were synthesized to have polycationic, polyanionic or neutral charges. Two human cell lines (A431 epidermoid carcinoma cells and EA.hy926 hybrid endothelial cells) were studied and the cellular uptake of c _e6 delivered by the conjugates of varying charge and free c _e6 was measured at conjugate c _e6 equivalent concentrations up to 0.4 μM. Uptake was time and concentration dependent and temperature dependent in the case of neutral and anionic conjugates. Relative uptake at 6 h for A431 cells was 73:15:4:1 and for EA. hy926 cells was 63:11:3:1 for cationic, anionic, neutral and free c _e6, respectively, but EA. hy926 cells took up 1.5-2 times as much c _e6 from all the conjugates as A431 cells. Localization as studied by fluorescence microscopy indicated that the cationic conjugate was in aggregates bound to the plasma membrane, while the other forms were internalized in organelles and membranes. Phototoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetra-zolium bromide (MTT) assay after irradiation with5–20 J cm⁻²of 666 nm light. In contrast to the uptake, the order of phototoxicity for both cell types per mole of c _e6 uptake per cell was neutral ≫ anionic > cationic > free c _e6. Polymeric c _e6 conjugates bearing positive, negative and neutral charges may have different tissue-localizing properties and could play a role in photodynamic therapy.     

ACTION SPECTRA FOR THE GENERATION OF SINGLET OXYGEN FROM MITOCHONDRIAL MEMBRANES FROM SOYBEAN (Glycine max) HYPOCOTYLS

Abstract— The action spectrum for the generation of singlet oxygen (¹O₂) from mitochondrial membranes under aerobic conditions was measured at wavelengths between 360 and 600 nm, using sub-mitochondrial particles (SMP) prepared from soybean hypocotyls. The spectrum, showing a peak at about 420 nm, remarkably resembles the absorption spectra of the Fe-S centers of nonheme iron proteins. Disruption of the Fe-S centers by treating SMP with mersalyl acid resulted in a substantial decrease in the efficiency of ¹O₂ generation, leaving an action spectrum whose pattern is significantly similar to the absorption spectrum of flavins, at least in the region of near UV and blue light wavelengths. Estimating the contribution of the Fe-S centers to the generation of ¹O₂ from SMP, we suggest that the Fe-S centers act as very important endogenous photosensitizers in plant cells, in so far as the type II mechanism is concerned. Possible involvement of mitochondrial flavoproteins in the generation of ¹O₂ is also discussed.     

INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.     

Photoactivated Psoralens Elicit Defense Genes and Phytoalexin Production in the Pea Plant

In the pea plant ( Pisum sativum ), compounds that intercalate into DNA induce the production of ∼20 major proteins similar to the pattern induced during nonhost disease resistance to the bean fungal pathogen, Fusarium solani f.sp. phaseoli . The pea phytoalexin, pisatin, as well as RNA homologous to several disease-resistance response (DRR) genes accumulate following treatment with these compounds. Psoralen was chosen to characterize this interaction further because it intercalates into DNA and, following irradiation with 365 nm UV light (UV₃₆₅), forms covalent bonds with pyrimidines on either or both strands of DNA. This produces monoadducts or cross-links, respectively. Dose experiments showed that 60 μg/mL 4'-aminomethyl-4,5',8-trimethylpsoralen followed by 18 J/cm² UV₃₆₅ was sufficient to produce an accumulation of pisatin similar to that produced in response to the fungus. Under these inducing conditions, there was an average of 0.19 adducts per kb of pea genomic DNA. The accumulation of pisatin and the RNA of several DRR genes by psoralen required photoactivation, which suggests that covalent binding to DNA was necessary for induction. As the promoters of several putative fungal-induced pea genes contain long stretches of d(AT)_n, which is the preferred psoralen photobinding site, restriction fragments spanning DRR genes were examined after in vivo psoralen treatment. The rate of crosslinking was compared between fungal-induced and noninduced genes using a modified Southern blot analysis. Implications of the induction of the DRR due to psoralen binding are discussed.     

In vitro Fluence Rate Effects in Photodynamic Reactions with AIPcS4 as Sensitizer

Abstract— It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AlPcS₄) is more efficient in photosensi-tization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m² as compared to 500 W/m². Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo , does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explanation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.     

TIME RESOLVED STUDIES OF 1.27 μm LUMINESCENCE FROM SINGLET OXYGEN GENERATED IN HOMOGENEOUS and MICROHETEROGENEOUS FLUIDS

-The luminescence at 1.27 μm from the ³→→¹δ_g transition of the oxygen molecule has been detected from a variety of liquid systems. A Q-switched laser delivering pulses of 532 nm light was the excitation source, a germanium photodiode was the detector and substituted porphyrins were used as photosensitizers. Protio- and deutero- forms of several solvents were studied and the singlet oxygen lifetimes determined directly agreed well with published values. T_δ in D₂O was found to be 55 μs and, by extrapolation from a series of H₂O - D₂O mixtures, a value of 3.3 μs was obtained for T_δ in H₂O. The technique was shown to be useful in measuring T_δ values in several microheterogeneous systems such as surfactant micelles, vesicles and human serum albumin.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

Rapid Initiation of Apoptosis by Photodynamic Therapy

Abstract— Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD₅₀), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer antagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucle-osomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD₅₀ PDT doses. Photodamage from two cat-ionic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD₁₀, LD₅₀ or LD₉₅ PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may therefore be a major determinant of the initiation and rate of progression of apoptosis.     

THE EFFECT OF PORPHYRIN PHOTOSENSITIZERS ON THE TOXICITY OF l,3-BIS(2-CHLOROETHYL)-1- NITROSOUREA IN C57BL/6J MICE

Abstract The most widely used agents for photodynamic therapy are the porphyrin photosensitizers. It has been shown that hematoporphyrin derivative (HpD) can cause murine marrow hypercellularity and splenic hypertrophy. We have examined the effect on survival and marrow cellularity of high dose l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) after HpD or dihematoporphyrin ether (DHE) pretreatment in C57BL/6J mice.
The lethal toxicity of the LD_S0+ 10% dose of BCNU (60 mg kg⁻¹) was significantly reduced by pretreatment with HpD when the HpD was administered at least 3 days prior to the BCNU. HpD administered 1 or 2 days prior to BCNU or after BCNU had no effect. The percent death rate was reduced from 80 to 0% when HpD was administered 7 and 5 days prior to BCNU.
No alteration of the lethal toxicity rate of BCNU at doses of 80 mg kg⁻¹ were identified with DHE pretreatment although some increase in median survival was noted in two groups. Some reduction in lethal toxicity was noted when 60 mg kg⁻¹ BCNU was used and the pretreatment dose of DHE was 10 or 25 mg kg⁻¹ given twice 3 days apart. Furthermore, a significant reduction of BCNU induced marrow cell depletion was found when low doses of DHE were used as pretreatment. High doses of DHE resulted in marrow depletion. Both HpD and DHE altered the toxicity of BCNU.
Should porphyrin photosensitizers, which alone have little toxicity, prove to protect against nitrosourea toxicity then an important dose limiting factor (myelotoxicity) could be altered if not reduction in the tumouricidal activity occurs.     

Synthesis and characterization of two Ni(II) complexes with furfural s-methylthiosemicarbazone

Synthesis of two Ni(II) complexes with furfural S-methylthiosemicarbazone (HL) of the formula [Ni(HL)₂(H₂O)₂](ClO₄)₂ (A) and [Ni(HL)₂(ClO₄)₂] (B) are reported. Compound A was obtained from an ethanolic solution of Ni(ClO₄)₂·6H₂O and HL, whilst compound B was produced by heating compound A to 378 K. An X-ray analysis of the complex A showed that it has a trans(H₂O)-trans(HL) octahedral configuration in which HL behaves as a bidentate (NN) ligand. On the basis of the IR and electronic spectra as well as the magnetism, it was found that the compound B has also an octahedral configuration in which, HL and ClO₄ groups, are coordinated.     

TEMPORAL and SPECTRAL SEPARATION OF SINGLET OXYGEN LUMINESCENCE FROM NEAR INFRARED EMITTING PHOTOSENSITIZERS

Abstract— The luminescence emission of singlet molecular oxygen (¹O₂) generated by bacteriopheophytin a, a near-infrared-emitting photosensitizer, was measured using a new high-sensitivity spectrometer system for time- and spectral-resolved near-infrared detection. The instrument uses a low energy pulsed nitrogen laser (40 μJ per pulse) to excite the photosensitizer optically and is capable of a time resolution of 40 ns per data point and an instrument response function of 350 ns FWHM (full width at half maximum). The use of a low-energy (and relatively low cost) source provides sufficient system sensitivity to measure time-resolved spectra in the near infrared with high spectral and temporal resolution. The simultaneous detection, with high accuracy and repeatability, of both the temporal and spectral dependence of the photoprocesses of ¹O₂ generation, especially with near-infrared-emitting photosensitizers, may further stimulate the current intensive investigations concerning the activity of ¹O₂ to biomolecules.