T.M.A.R. Dubbelman

Photodynamic effects of protoporphyrin on human erythrocytes. Nature of the cross-linking of membrane proteins.

Protoporphyrin-sensitized photooxidation in human red blood cell membranes leads to severe deterioration of membrane structure and function. The membrane damage is caused by direct oxidation of amino acid residues, with subsequent cross-linking of membrane proteins. The chemical nature of these cross-links was studied in model systems, isolated spectrin and red cell ghosts. Cysteine and methionine are not involved in the cross-linking reaction. Further it could be shown that dityrosine formation, the crucial mechanism in oxidative cross-linking of proteins by peroxidase-H2O2 treatment, plays no role in photodynamic cross-linking. Experimental evidence indicated that a secondary reaction between free amino groups and a photooxidation product of histidine, tyrosine or tryptophan is involved in photodynamic cross-linking. This was deduced from the reaction observed between compounds containing a free amino group and photooxidation products of these amino acids, both in model systems, isolated spectrin and erythrocyte ghosts. In accordance, succinylation of free amino groups of membrane proteins or addition of compounds with free amino groups protected against cross-linking. Quantitative data and consideration of the reaction mechanisms of photodynamic oxidation of amino acids make it highly probable that an oxidation product of histidine rather than of tyrosine or tryptophan is involved in the cross-linking reaction, via a nucleophilic addition by free amino groups.

Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals

The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.

Fundamentals of photodynamic therapy: cellular and biochemical aspects

Photodynamic therapy of cancer is based on the photosensitizing ability of dyes which, after administration, are present in a somewhat higher concentration in tumors than in surrounding normal tissue. After light activation of the sensitizer, singlet oxygen and probably oxygen free radicals are formed and consequently all kinds of cellular components are affected. This review focuses on cellular and biochemical aspects of photodynamic therapy. Both damage to different cellular targets and cellular responses after photodynamic treatment are discussed.

Peroxide-induced membrane damage in human erythrocytes

Erythrocytes exposed to H2O2 or t-butyl hydroperoxide (tBHP) exhibited lipid peroxidation and increased passive cation permeability. In the case of tBHP a virtually complete inhibition of both processes was caused by butylated hydroxytoluene (BHT), whereas pretreatment of the cells with CO increased both lipid peroxidation and K+ leakage. In the experiments with H2O2, on the other hand, both BHT and CO strongly inhibited lipid peroxidation, without affecting the increased passive cation permeability. These observations indicate different mechanisms of oxidative damage, induced by H2O2 and tBHP, respectively. The SH-reagent diamide strongly inhibited H2O2-induced K+ leakage, indicating the involvement of SH oxidation in this process. With tBHP, on the contrary, K+ leakage was not significantly influenced by diamide. Thiourea inhibited tBHP-induced K+ leakage, without affecting lipid peroxidation. Together with other experimental evidence this contradicts a rigorous interdependence of tBHP-induced lipid peroxidation and K+ leakage.

Inhibition of enzymes and oxidative damage of red blood cells induced by t-butylhydroperoxide-derived radicals

The effects of t-butylhydroperoxide (tBHP), its alkoxyl radical (tBuO.) and its peroxyl radical (tBuOO.) in model systems and on red blood cells were studied. Glyceraldehyde-3-phosphate dehydrogenase was strongly inhibited by tBHP via a direct reaction of the hydroperoxide with an essential sulfhydryl group in the enzyme molecule. Several other enzymes were unaffected by tBHP. Alcohol dehydrogenase was strongly inhibited by tBuO. but was much less sensitive to tBuOO.. Lysozyme, lactate dehydrogenase and trypsin, on the other hand, were very sensitive to the peroxyl and not, or much less, to the alkoxyl radical, whereas acetylcholinesterase was very sensitive to both radicals. tBuOO. caused covalent binding of tryptophan, tyrosine, histidine and methionine to serum albumin. The corresponding alkoxyl radical was ineffective in this respect. Conversely, tBuO. caused peroxidation of linolenic acid, whereas tBuOO. did not. Incubation of human erythrocytes with tBHP caused lipid peroxidation and K+ leakage. Both effects were caused by tBHP-derived radicals generated in a reaction of the hydroperoxide with hemoglobin. With radical scavengers it was possible to dissociate tBHP-induced lipid peroxidation and K+ leakage, demonstrating that these two processes are not causally related. Experimental results indicate that tBuO. causes lipid peroxidation, whereas tBuOO. is responsible for K+ leakage.

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.

Protoporphyrin-induced photodynamic effects on transport processes across the membrane of human erythrocytes.

Previous studies have shown that illumination of erythrocytes with visible light in the presence of protoporphyrin results in cross-linking of membrane proteins and deterioration of several membrane functions, e.g. active transport of K+ and Na+. In the present study it is shown that carrier-mediated transport of glucose, L-leucine, sulphate and glycerol is also inhibited by the photodynamic process, whereas non-specific permeability of glycerol and thiourea is increased. It is shown that these effects are not caused by lipid peroxidation, but by photooxidation of membrane proteins. The inhibition of carrier-mediated transport is caused either by photodynamic oxidation of susceptible essential amino acid residues of the carrier molecules, or by an aspectific perturbation of the membrane structure, leading to inhibition of carrier functions.

Photodynamic effects of hematoporphyrin-derivative on transmembrane transport systems of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative causes deterioration of various membrane functions. Most sensitive to photodynamic inactivation are the energy-coupled transport systems for aminoisobutyric acid and for Rb+. The facilitated diffusion system for 2-deoxy-D-glucose is slightly less sensitive. After longer illumination periods also the membrane barrier function is impaired, as reflected by K+ leakage and increased passive Rb+ uptake. After still longer illumination periods intermolecular protein crosslinking can be observed. This makes it unlikely that intermolecular protein crosslinking is causally involved in the deterioration of these membrane functions.

The characterisation of three substituted zinc phthalocyanines of differing charge for use in photodynamic therapy. A comparative study of their aggregation and photosensitising ability in relation to mTHPC and polyhaematoporphyrin

Three substituted zinc (II) phthalocyanines (one anionic, one cationic and one hydrophobic) have been compared to two clinically used photosensitisers, 5,10,15,20-tetra (m-hydroxyphenyl) chlorin (mTHPC) and polyhaematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Oxygen-consumption experiments, performed to follow the photo-oxidation of tryptophan, histidine and bovine serum albumin (BSA), suggest that the anionic phthalocyanine is the most efficient photosensitiser. The efficacy of BSA oxidation is much greater than that of tryptophan or histidine, which is partly due to monomerisation of the sensitisers upon binding to BSA. Spectra recorded in aqueous solution reveal that all five compounds are highly aggregated, but monomerisation is induced upon the addition of BSA or methanol. Using a range of methanol-buffer solutions, the aggregation state has been directly related to the efficacy of tryptophan photo-oxidation with maximal rates of oxidation achieved when the sensitiser is monomeric. Using erythrocytes as a simple membrane model, the efficacy of each sensitiser exhibits a different trend from that predicted by oxygen-consumption experiments. The anionic phthalocyanine is the least effective at photohaemolysis, whereas the cationic and hydrophobic phthalocyanines have improved activity over PHP and mTHPC.

Protoporphyrin-sensitized photodynamic modification of proteins in isolated human red blood cell membranes.

Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross‐linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis.