PHTHALOCYANINE-INDUCED PHOTOHEMOLYSIS: STRUCTURE-ACTIVITY RELATIONSHIP AND THE EFFECT OF FLUORIDE

Abstract— Phthalocyanine (Pc) containing AI, Ga or Zn as central metal ligand and substituted with a varying number of sulfonic acid residues as well as additional benzene rings were synthesized and their photodynamic activity was assayed using photohemolysis of human erythrocytes as an endpoint. The Pc derivatives vaned > 300-fold in their photodynamic activity. Activity corrclated with binding of the dye to the cell, with the exception of some of the amphiphilic dyes where cell uptake was an order of magnitude higher than expected from the observed activity. Fluoride was shown to inhibit AIPcS_n-induccd photohemolysis. This effect occurred also with other AlPc and GaPc derivatives, but the concentration of F required to slow photohemolysis by a factor of two (K_i) varied between 4 μ M and 10 mM. Fluorescence spectral studies indicated complex formation between F⁻ and the dye, which was stronger for AlPc than GaPc derivatives. Ultrastructural studies using scanning electron microscopy showed that the photosensitized cells were converted to spherocytes and that F⁻ prevented this to a large extent.     

MEASUREMENT OF DNA CROSSLINKS BY S1 NUCLEASE: INDUCTION AND REPAIR IN PSORALEN-PLUS-360 nm LIGHT TREATED ESCHERICHIA COLI

Abstract—DNA crosslinks in Escherichia coli cells. exposed to 4.5',8-trimethylpsoralen plus 360 nm light, were measured using a rapid and sensitive new approach. The assay is based on the specificity of S₁ nuclease from Aspergillus oryzae to single-stranded DNA. Bacterial cells were lysed and the DNA denatured by alkali. Following acid neutralization. crosslinked DNA undergoes spontaneous renaturation and is rendered S₁-nuclease resistant and therefore acid-precipitable. The single-stranded fraction after denaturation by alkali decreases with increasing near UV light exposure in the presence of TMP following first order kinetics. The kinetics were faster when exposure was at 4°C rather than at 20°C. This suggests that excision of crosslinks occurs during exposure at the higher temperature. Indeed. since the rate of DNA crosslinking in a uvr B mutant which is excision-deficient was higher than in wild type bacteria at 4°C, some excision must have occurred even in the cold. DNA from excision-proficient cells incubated at 37°C following exposure to TMP-plus-near UV at 4° showed a greater single stranded fraction than that from non-incubated cells. This indicates repair of DNA crosslinks. which proceeded with a half-time of 8 min at 37°C and was unaffected by substitution of thymine in DNA by 5-bromouracil.     

Computation in gene networks

Genetic regulatory networks have the complex task of controlling all aspects of life. Using a model of gene expression by piecewise linear differential equations we show that this process can be considered as a process of computation. This is demonstrated by showing that this model can simulate memory bounded Turing machines. The simulation is robust with respect to perturbations of the system, an important property for both analog computers and biological systems. Robustness is achieved using a condition that ensures that the model equations, that are generally chaotic, follow a predictable dynamics.     

THE EFFECT OF FLUORIDE ON BINDING and PHOTODYNAMIC ACTION OF PHTHALOCYANINES WITH PROTEINS

Fluoride inhibits chloroaluminum phthalocyanine tetrasulfonate (AlPcS)-induced photohemolysis when added to dye loaded cells prior to light exposure. The mechanism by which F- exerts this effect was studied by measuring the binding of phthalocyanine (Pc) to various proteins in the absence and presence of F-. Parallel measurements were made of the photodynamic action under these conditions. Fluoride reduced the binding to proteins of AlPcS and CoPcS. The binding of CuPcS, ZnPcS and H2PcS was not affected. When bound to bovine serum albumin and exposed to light, H2Pc, ZnPc and AlPcCl were bleached at a biphasic rate. Only the photobleaching of AlPcCl was affected by F-. The effect of F- was to inhibit the initial rapid phase without affecting the slower phase. In the presence of D2O only the second phase of photobleaching was enhanced, in the absence or presence of F-. No effect of F- was observed on tryptophan photooxidation or glyceraldehyde-3-phosphate dehydrogenase photoinactivation by AlPcS. Crosslinking of spectrin monomers photosensitized by AlPcS was inhibited by F- in parallel with the reduced binding of dye to the protein. It is concluded that F- exerts its effect by complexing with metal ligands of Pc. As a result, the dye may be released from the protein or the binding mode may be changed in such a way that effective photochemistry is prevented. Primary photophysical processes of Pc most probably are not affected by F-.     

Structure-Activity and Mechanism Studies on Silicon Phthalocyanines with Plasmodium falciparum in the Dark and Under Red Light

Syntheses for the new photosensitizers HOSiPc-OSi(CH₃)₂(CH₂)₃N(CH₂)₁ or ₃(CH₃)₂, Pc 34 and Pc 25, have been developed and the order of activity of these photosensitizers and the previously reported photosensi-tizer Pc 4, HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂, in the dark and with broad-band red light toward Plasmodium falciparum in red blood cell (RBC) suspensions has been studied. The order of activity has been found to be Pc 4 Pc 34 Pc 25. Thus, the activity of the photosensitizers under both sets of conditions is inversely proportional to the length of their terminal amino alkyl chains. The 50% inhibition dye concentration (IC₅₀) in the dark for the parasites in RBC suspension with Pc 4 is 24 nM and the dye concentration and light fluence that yield:3 log₁₀ of parasite inactivation with Pc 4 are 2 mM and 3 J/cm², respectively. The synthesis of DNA and proteins by the parasites in culture was strongly inhibited by Pc 4 in the dark while parasite lactate dehydrogenase (pLDH) activity was unaffected. With Pc 4 and light, DNA and protein synthesis of the parasites in culture was strongly inhibited, pLDH activity of the parasites was moderately inhibited and ribosome density of the parasite cells was reduced. Gel electrophoresis studies showed that synthesis of all parasite proteins was inhibited to a similar extent. These results suggest that Pc 4 both in the dark and with light inactivates the cells by disturbing their machinery for the synthesis of not just one but a whole series of proteins. It is concluded that Pc 4 and light may be able to serve as a practical sterilization combination not only for HIV and other viruses but also for malaria parasites in RBC concentrates, and that Pc 4 by itself may have potential as a chemotherapeutic agent toward malaria.     

RECOVERY FROM INHIBITION BY UV-IRRADIATION OF ORNITHINE DECARBOXYLASE INDUCTION IN HUMAN CELLS: IMPLICATION OF EXCISION REPAIR

Abstract— Exposure of stationary-phase human breast carcinoma(T–47D) cells to far-UV light (254 nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (D₄= 2 J m_-2) followed by an exponential decline (D₀= 4.2 Jm_-2). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for20–24 h after irradiation. Hydroxyurea (HU) when present at 3 mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50 nM of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, we interpret the above results to mean that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.     

Photosensitization of Chinese hamster cells by water-soluble phthalocyanines

Abstract— Phthalocyanines are efficient photosensitizers of cultured mammalian cells and are considered for use in photodynamic therapy. The photobiological properties of chloroaluminum phthalocyanine sulfonate (AIPCS) were compared to those of the unsubstituted, water-insoluble derivative (AIPC). The development of photosensitization after addition of the dye into growth medium is ca. 8 times more rapid for AlPC than for AIPCS. Conversely, the loss of photosensitization when cells are incubated in a dye-free growth medium following a period of dye uptake, is also faster for AIPC. The dye uptake followed a kinetic behavior similar to the development of photosensitivity, but the loss of dye was too slow for both AlPC and AIPCS to explain loss of photosensitivity. When cells are incubated prior to illumination with AIPCS in phosphate buffered saline instead of growth medium, shorter time and smaller amount of dye are required to achieve the same level of photosensitization. The dependence of photosensitivity on dye concentration is linear for both AIPC and AIPCS. As already found for AIPC, photosensitization by the water-soluble derivative is also not enhanced in D₂O, suggesting that singlet oxygen is not involved in the cytotoxic response. Sodium salicylate, which was found to enhance the effect of AlPC was also effective with AIPCS. This effect is quite specific since the meta and para isomers had no effect. The metal atom complexed with the phthalocyanine ring is significant for the photobiological activity. Among the compounds tested, those containing Al or Zn are most active.     

PHTHALOCYANINE PHOTOSENSITIZATION OF MAMMALIAN CELLS: BIOCHEMICAL and ULTRASTRUCTURAL EFFECTS

Abstract— The concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺ and CI⁻ ions in the cytoplasm of octopus photoreceptor cells were determined before and after illumination by electron probe X-ray microanalysis. The concentrations of these elements in the dark-adapted photoreceptor cells were: Na⁺, 68.4; K⁺, 111.4; Ca²⁺, 4.0; Mg²⁺, 16.4; and CI⁻, 102.9 m M /kg of cytoplasm. Illumination raised the concentration of Na⁺ by 58 m M and that of Cl⁻ by 23 m M and reduced the K⁺ concentration by 47 m M /kg of cytoplasm. A trace increase of intracellular Ca²⁺ and a trace decrease of Mg²⁺ were observed. These results confirm the hypothesis that sodium ions flow in on illumination, and suggest the influx of chloride ions and the outflux of potassium ions during illumination. The intracellular concentrations of Na⁺, K⁺ and Cl⁺ can give the basis for calculating the ion permeability of ion channels in octopus photoreceptor cell membranes, using values of the membrane potentials obtained by electrophysiological studies     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.