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Papers by Tom Dubbelman
Inborn Errors of Metabolism in Man
Biochemical Journal, 1993
The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid me... more The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid metabolism and its relation to clonogenicity have been studied in human bladder-tumour cells. Photodynamic treatment resulted in a transient release of arachidonic acid-derived compounds; prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) especially were strongly increased. This release was reduced by chelation of intracellular Ca2+ with Quin-2 or by lowering the extracellular Ca2+ concentration in the medium with EGTA, presumably resulting in inhibition of phospholipase A2. A similar reduction was obtained when indomethacin, an inhibitor of the cyclo-oxygenase pathway, was added prior to light exposure. These three treatments enhanced the photosensitivity, as revealed by the clonogenicity assay. Incubation with PGE2 prior to light exposure, but not with TXB2, protected against reproductive-cell death. The results of these experiments suggest that Ca(2+)-mediated activation of cyclo-oxygena...
Recent Research Developments in Photochemistry and Photobiology, 2000
The inactivation of viruses in cellular blood products is a relatively new application of photody... more The inactivation of viruses in cellular blood products is a relatively new application of photodynamic treatment. This reviews will give an overview of results obtained on this subject at the departments of Molecular Cell Biology and Immunohematology & Bloodbank, Leiden University Medical Center, Leiden, The Netherlands. The crucial factor in the sterilization of blood products is the quality of the different cellular components. Therefore an extensive study into the shelf-life of photodynamically-treated red blood cell suspensions was performed. Also variations in the treatment and storage protocol were evaluated for their contribution to the preservation of the red blood cells. Besides excisting protocols, also a new series of photosensitizers was assayed. Two sensitizers from this series, nicknamed Sylsens A and B, showed efficient virucidal activity with only very limited damage to red blood cells. Since RBCC contain a certain number of leukocytes, which are involved in the immunomodulative effects of blood transfusions, it is important to assess the effect of photodynamic treatment on these cells. Therefore, the viability and the mechanism of cell death of the various leukocyte subpopulations after virucidal treatment with different photosensitizers were determined. It has been shown that both the efficacy and the specificity of a photodynamic treatment can be influenced by the fluence rate used during illumination. A photochemical study into this phenomenon and its potential use in sterilization of RBCC was performed. For different reasons, identification of the viral targets for photodynamic treatment is important: it will contribute to the search for optimal inactivation conditions with respect to sensitizer properties and illumination protocols, and furthermore it is necessary for validation of the inactivation protocol. The primary targets for the inactivation of the lipid-enveloped Vesicular Stomatitis Virus by a hydrophilic and a hydrophobic photosensitizer were determined.
Biochemical Journal, 1991
Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a confo... more Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a conformational mechanism, without involvement of covalent reactions. On the other hand, photodynamic inactivation of the enzyme (induced by illumination in the presence of Photofrin II) was caused by photo-oxidation of the essential thiol group in the active centre. A short photodynamic treatment of the enzyme, leading to only a limited inactivation, caused a pronounced potentiation of subsequent thermal inactivation, as measured over the temperature range 40-50 degrees C. Analysis of the experimental results according to the Arrhenius equation revealed that both the activation energy of thermal inactivation and the frequency factor (the proportionality constant) were significantly decreased by the preceding photodynamic treatment. The experimental results indicate a mechanism in which limited photodynamic treatment induced a conformational change of the protein molecule. This conformational ...
Biochemical Journal, 1991
Heat treatment of human erythrocytes led to increased passive cation permeability, followed by ha... more Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4′-di-isothiocyano-2,2′-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakag...
Biochemical Journal, 1987
Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enz... more Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell...
British Journal of Cancer, 1993
Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in... more Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent 'sandwich' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-' BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascularand tissue damage after a therapeutic light dose (760 nm, 600 J cm-2) was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT (20mg kg-', 760 nm, 100 J cm-2) h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, I h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shutdown was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm and occlusion probably due to thrombi formation. Oedema was notable, especially following PDT with 600Jcm-2 at 24h p.i.
Light in Biology and Medicine, 1988
Although it is well known that cells do not survive photodynamic treatment with photosensitizers,... more Although it is well known that cells do not survive photodynamic treatment with photosensitizers, the actual mechanism of cell death is unknown in most cases and may also be different in different cells. Most cellular systems prove to be sensitive to photodynamic damage, because most constituents of cells, proteins, nucleic acids, lipids and coenzymes can in principle be photooxidized. The actual oxidation depends on many factors such as the intracellular localization of both the photosensitizer and the target molecules. An example is the intralysosomal enzyme β-glucuronidase, which in intact L929 cells is not inactivated at all, but when the cells are disrupted by sonication this enzyme proves to be quite sensitive to photoinactivation by HPD (Boegheim et al., 1987). The mechanism of inactivation of cellular systems has only been clarified in very few cases. Photooxidation of cysteine or histidine residues is often the reason for the inactivation of functional proteins. Examples are the inactivation of the enzymes glyceraldehyde-3-phosphate dehydrogenase and succinate dehydrogenase and the inhibition of some membrane transport systems.
Photochemotherapy: Photodynamic Therapy and Other Modalities, 1996
ABSTRACT
Photochemistry and Photobiology, 1991
... reticulum in the dark (Abramson et al., 1988) and that oxidation of protein sulfhydryl groups... more ... reticulum in the dark (Abramson et al., 1988) and that oxidation of protein sulfhydryl groups by toxic agents inhibits ATP-dependent Ca" sequestration by rat liver microsomes (Thor et a]., 1985). ...Ben-Hur, E. (1991) Basic photobiology and mechanisms of action of phthalocyanines ...
Lasers in Medical Science, 1993
Female WAG/RIJ rats with isologous RMA-mammary or rhabdomyosarcoma tumours transplanted subcutane... more Female WAG/RIJ rats with isologous RMA-mammary or rhabdomyosarcoma tumours transplanted subcutaneously on the flank and thigh were given 10mg kg 1 bacteriochlorin a intravenously (IV). In vivo fluorescence properties of this new photosensitizer were studied in a first attempt by using intensified fluorescence imaging. Analysis of the digitized images yielded tumour/ muscle fluorescence ratios. Thirty minutes after IV injection a ratio of about 2.4 was reached, which was maintained for at least 48 h.
Journal of Photochemistry and Photobiology B: Biology, 1995
To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich envir... more To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich environment, the photosensitizing efficacy was tested by clonogenic survival of Chinese hamster ovary and T24 (human bladder carcinoma) cells. Confluent cell layers were incubated with 2.5 tzg m1-1 BCA in cell culture medium for 1, 4, 6, 18 and 24 h. Upon illumination with red light it was found that BCA was not effective as a photosensitizer in this medium. Extraction methods showed that this lack of photosensitization could not be explained by the inability of the dye to enter the cells in the presence of cell culture medium. The presence of cell culture medium did not change the spectral properties of BCA to an appreciable extent. Standard KBr density gradient ultracentrifugation showed that in the presence of cell culture medium approximately 20% of the BCA was sedimented with low density lipoprotein (LDL) and 60% with high density lipoprotein (HDL). Incubating T24 cells 18 h before the clonogenic cell survival assay in serum-deficient medium restored the photosensitizing properties of BCA. It is proposed that in a protein-rich (in vivo) environment BCA associates with lipoproteins and can be taken up by malignant neoplasms via the LDL pathway.
Journal of Photochemistry and Photobiology B: Biology, 1999
l luman adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid ... more l luman adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal call' ~+erum. The treatment induces a linear accumulation of protoporphyrin IX (PplX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PplX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphatebuffered saline (30%). Exposure to white light at an intensity of 30 W/m 2 for 18 min results in 95% reduction of clonogenicity in cells treated witil 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate deh ydrogenase) and lysosomes (acid phosphatase and [3-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by lhe same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rho, lamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces dan~age to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.
Journal of Photochemistry and Photobiology B: Biology, 1989
International Journal of Radiation Biology, 1993
Bacteriochlorin a (BCA), a new photosensitizer for photodynamic therapy, was labelled with 99mTc-... more Bacteriochlorin a (BCA), a new photosensitizer for photodynamic therapy, was labelled with 99mTc-pertechnetate following a method for the irreversible coupling of 99mTc-pertechnetate to proteins. Biodistribution studies were conducted in male Syrian Golden hamsters with hamster Greene melanoma implanted s.c. on both sides of the abdomen. After i.v. administration of 99mTc-pertechnetate-labelled BCA 17 tissue and fluid samples were analysed at time intervals ranging from 1 to 24 h. Technetium-labelled BCA showed a pronounced affinity for tissues belonging to the reticuloendothelial system. Peak activities, 1 h post-injection, were distributed as follows: lung, liver, spleen, urine > small intestine, kidney, blood, heart, stomach, large intestine > thyroid, tumour, bone, skin, muscle, eye >> brain. It is concluded that the technetium-labelled photosensitizer BCA does not accumulate selectively in neoplastic tissue.
Cellular and Molecular Life Sciences, 1997
The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular pac... more The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1984
ABSTRACT
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-... more 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
Chinese hamster ovary (CHO) cells and 'I'24 human bladder transitional carcinoma cells were treat... more Chinese hamster ovary (CHO) cells and 'I'24 human bladder transitional carcinoma cells were treated with the photoscnsitizers alum;bum phthalocyanine (AIPe) 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 rain after exposure and then returning to basal level (~ 200 nM). The level of the peak [Ca2+]i depended on the light flucnce, 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 AIPc and with HPD-PDT of T24 cells extracellular Ca 2+ 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., a20 2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.
Porphyrins in Tumor Phototherapy, 1984
Porphyrin photoradiation therapy for the treatment of various malignant tumors is a recent rapidl... more Porphyrin photoradiation therapy for the treatment of various malignant tumors is a recent rapidly developing technique and progress has been fast and promising1,2). The principle of porphyrin photoradiation therapy is relatively simple. Following systematic administration, porphyrins, especially the so-called hematoporphyrin-derivative (HpD) are accumulated to higher concentrations in malignant than in normal cells3). During subsequent exposure of the cells to visible light the accumulated porphyrin acts as photodynamic sensitizer 4) and causes oxidation of cellular constituent presumably by formation of singlet oxygen and possibly hydroxyl radicals. These oxidations lead to disturbed functions and, ultimately, cell death. Three mechanisms may be responsible e.g. membrane deterioration, DNA-damage and photodynamic inactivation of crucial enzyme systems. Studies of Kessel et al. indicate that membrane damage may be the direct cause of photodynamic cell death in tumors 5–7). Illumination of L1210 and SS-1 cells incubated with porphyrin leads to cross-linking of membrane proteins 6) as described before with red cell membranes 8,9). Further,loss of cell viability was correlated with inhibition of transmembrane cycloleucine transport 5).
Inborn Errors of Metabolism in Man
Biochemical Journal, 1993
The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid me... more The effects of haematoporphyrin-derivative-mediated photodynamic treatment on arachidonic acid metabolism and its relation to clonogenicity have been studied in human bladder-tumour cells. Photodynamic treatment resulted in a transient release of arachidonic acid-derived compounds; prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) especially were strongly increased. This release was reduced by chelation of intracellular Ca2+ with Quin-2 or by lowering the extracellular Ca2+ concentration in the medium with EGTA, presumably resulting in inhibition of phospholipase A2. A similar reduction was obtained when indomethacin, an inhibitor of the cyclo-oxygenase pathway, was added prior to light exposure. These three treatments enhanced the photosensitivity, as revealed by the clonogenicity assay. Incubation with PGE2 prior to light exposure, but not with TXB2, protected against reproductive-cell death. The results of these experiments suggest that Ca(2+)-mediated activation of cyclo-oxygena...
Recent Research Developments in Photochemistry and Photobiology, 2000
The inactivation of viruses in cellular blood products is a relatively new application of photody... more The inactivation of viruses in cellular blood products is a relatively new application of photodynamic treatment. This reviews will give an overview of results obtained on this subject at the departments of Molecular Cell Biology and Immunohematology & Bloodbank, Leiden University Medical Center, Leiden, The Netherlands. The crucial factor in the sterilization of blood products is the quality of the different cellular components. Therefore an extensive study into the shelf-life of photodynamically-treated red blood cell suspensions was performed. Also variations in the treatment and storage protocol were evaluated for their contribution to the preservation of the red blood cells. Besides excisting protocols, also a new series of photosensitizers was assayed. Two sensitizers from this series, nicknamed Sylsens A and B, showed efficient virucidal activity with only very limited damage to red blood cells. Since RBCC contain a certain number of leukocytes, which are involved in the immunomodulative effects of blood transfusions, it is important to assess the effect of photodynamic treatment on these cells. Therefore, the viability and the mechanism of cell death of the various leukocyte subpopulations after virucidal treatment with different photosensitizers were determined. It has been shown that both the efficacy and the specificity of a photodynamic treatment can be influenced by the fluence rate used during illumination. A photochemical study into this phenomenon and its potential use in sterilization of RBCC was performed. For different reasons, identification of the viral targets for photodynamic treatment is important: it will contribute to the search for optimal inactivation conditions with respect to sensitizer properties and illumination protocols, and furthermore it is necessary for validation of the inactivation protocol. The primary targets for the inactivation of the lipid-enveloped Vesicular Stomatitis Virus by a hydrophilic and a hydrophobic photosensitizer were determined.
Biochemical Journal, 1991
Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a confo... more Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a conformational mechanism, without involvement of covalent reactions. On the other hand, photodynamic inactivation of the enzyme (induced by illumination in the presence of Photofrin II) was caused by photo-oxidation of the essential thiol group in the active centre. A short photodynamic treatment of the enzyme, leading to only a limited inactivation, caused a pronounced potentiation of subsequent thermal inactivation, as measured over the temperature range 40-50 degrees C. Analysis of the experimental results according to the Arrhenius equation revealed that both the activation energy of thermal inactivation and the frequency factor (the proportionality constant) were significantly decreased by the preceding photodynamic treatment. The experimental results indicate a mechanism in which limited photodynamic treatment induced a conformational change of the protein molecule. This conformational ...
Biochemical Journal, 1991
Heat treatment of human erythrocytes led to increased passive cation permeability, followed by ha... more Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4′-di-isothiocyano-2,2′-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakag...
Biochemical Journal, 1987
Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enz... more Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell...
British Journal of Cancer, 1993
Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in... more Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent 'sandwich' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-' BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascularand tissue damage after a therapeutic light dose (760 nm, 600 J cm-2) was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT (20mg kg-', 760 nm, 100 J cm-2) h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, I h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shutdown was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm and occlusion probably due to thrombi formation. Oedema was notable, especially following PDT with 600Jcm-2 at 24h p.i.
Light in Biology and Medicine, 1988
Although it is well known that cells do not survive photodynamic treatment with photosensitizers,... more Although it is well known that cells do not survive photodynamic treatment with photosensitizers, the actual mechanism of cell death is unknown in most cases and may also be different in different cells. Most cellular systems prove to be sensitive to photodynamic damage, because most constituents of cells, proteins, nucleic acids, lipids and coenzymes can in principle be photooxidized. The actual oxidation depends on many factors such as the intracellular localization of both the photosensitizer and the target molecules. An example is the intralysosomal enzyme β-glucuronidase, which in intact L929 cells is not inactivated at all, but when the cells are disrupted by sonication this enzyme proves to be quite sensitive to photoinactivation by HPD (Boegheim et al., 1987). The mechanism of inactivation of cellular systems has only been clarified in very few cases. Photooxidation of cysteine or histidine residues is often the reason for the inactivation of functional proteins. Examples are the inactivation of the enzymes glyceraldehyde-3-phosphate dehydrogenase and succinate dehydrogenase and the inhibition of some membrane transport systems.
Photochemotherapy: Photodynamic Therapy and Other Modalities, 1996
ABSTRACT
Photochemistry and Photobiology, 1991
... reticulum in the dark (Abramson et al., 1988) and that oxidation of protein sulfhydryl groups... more ... reticulum in the dark (Abramson et al., 1988) and that oxidation of protein sulfhydryl groups by toxic agents inhibits ATP-dependent Ca" sequestration by rat liver microsomes (Thor et a]., 1985). ...Ben-Hur, E. (1991) Basic photobiology and mechanisms of action of phthalocyanines ...
Lasers in Medical Science, 1993
Female WAG/RIJ rats with isologous RMA-mammary or rhabdomyosarcoma tumours transplanted subcutane... more Female WAG/RIJ rats with isologous RMA-mammary or rhabdomyosarcoma tumours transplanted subcutaneously on the flank and thigh were given 10mg kg 1 bacteriochlorin a intravenously (IV). In vivo fluorescence properties of this new photosensitizer were studied in a first attempt by using intensified fluorescence imaging. Analysis of the digitized images yielded tumour/ muscle fluorescence ratios. Thirty minutes after IV injection a ratio of about 2.4 was reached, which was maintained for at least 48 h.
Journal of Photochemistry and Photobiology B: Biology, 1995
To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich envir... more To study the photosensitizing properties of bacteriochlorin a (BCA) in a (lipo)protein-rich environment, the photosensitizing efficacy was tested by clonogenic survival of Chinese hamster ovary and T24 (human bladder carcinoma) cells. Confluent cell layers were incubated with 2.5 tzg m1-1 BCA in cell culture medium for 1, 4, 6, 18 and 24 h. Upon illumination with red light it was found that BCA was not effective as a photosensitizer in this medium. Extraction methods showed that this lack of photosensitization could not be explained by the inability of the dye to enter the cells in the presence of cell culture medium. The presence of cell culture medium did not change the spectral properties of BCA to an appreciable extent. Standard KBr density gradient ultracentrifugation showed that in the presence of cell culture medium approximately 20% of the BCA was sedimented with low density lipoprotein (LDL) and 60% with high density lipoprotein (HDL). Incubating T24 cells 18 h before the clonogenic cell survival assay in serum-deficient medium restored the photosensitizing properties of BCA. It is proposed that in a protein-rich (in vivo) environment BCA associates with lipoproteins and can be taken up by malignant neoplasms via the LDL pathway.
Journal of Photochemistry and Photobiology B: Biology, 1999
l luman adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid ... more l luman adenocarcinoma cells of the line WiDr have been treated with 2 mM 5-aminolaevulinic acid (5-ALA) in the presence of 10% foetal call' ~+erum. The treatment induces a linear accumulation of protoporphyrin IX (PplX) for at least 7.5 h. After 7.5 h of incubation about 45% of the PplX accumulated is cell-bound, while the rest is found in the medium (25%) or lost from the cells during washing with phosphatebuffered saline (30%). Exposure to white light at an intensity of 30 W/m 2 for 18 min results in 95% reduction of clonogenicity in cells treated witil 2 mM 5-ALA for 3.5 h. The enzymatic activities of enzymes located in cytosol (glyceraldehyde 3-phosphate dehydrogenase and lactate deh ydrogenase) and lysosomes (acid phosphatase and [3-glucuronidase) are not influenced by a 5-ALA and light treatment inactivating about 35% of the cells. The MTT assay, which reflects mitochondrial dehydrogenase activity, but not succinate dehydrogenase, is partly inhibited by lhe same treatment. Treatment with 5-ALA in the absence of light increases O2 consumption by a factor of two, while the O2 consumption is inhibited when 5-ALA treatment is combined with exposure to light. In addition, 5-ALA and light exposure enhance accumulation of rho, lamine 123 by 40% and reduce the intracellular ATP level by 25%. Confocal laser scanning microscopical analysis indicates granular perinuclear localization of the PpIX formed by 5-ALA treatment. In conclusion, photodynamic treatment using 5-ALA as a prodrug induces dan~age to mitochondrial function without inhibiting lysosomal and cytosolic marker enzymes.
Journal of Photochemistry and Photobiology B: Biology, 1989
International Journal of Radiation Biology, 1993
Bacteriochlorin a (BCA), a new photosensitizer for photodynamic therapy, was labelled with 99mTc-... more Bacteriochlorin a (BCA), a new photosensitizer for photodynamic therapy, was labelled with 99mTc-pertechnetate following a method for the irreversible coupling of 99mTc-pertechnetate to proteins. Biodistribution studies were conducted in male Syrian Golden hamsters with hamster Greene melanoma implanted s.c. on both sides of the abdomen. After i.v. administration of 99mTc-pertechnetate-labelled BCA 17 tissue and fluid samples were analysed at time intervals ranging from 1 to 24 h. Technetium-labelled BCA showed a pronounced affinity for tissues belonging to the reticuloendothelial system. Peak activities, 1 h post-injection, were distributed as follows: lung, liver, spleen, urine > small intestine, kidney, blood, heart, stomach, large intestine > thyroid, tumour, bone, skin, muscle, eye >> brain. It is concluded that the technetium-labelled photosensitizer BCA does not accumulate selectively in neoplastic tissue.
Cellular and Molecular Life Sciences, 1997
The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular pac... more The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1984
ABSTRACT
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-... more 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
Chinese hamster ovary (CHO) cells and 'I'24 human bladder transitional carcinoma cells were treat... more Chinese hamster ovary (CHO) cells and 'I'24 human bladder transitional carcinoma cells were treated with the photoscnsitizers alum;bum phthalocyanine (AIPe) 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 rain after exposure and then returning to basal level (~ 200 nM). The level of the peak [Ca2+]i depended on the light flucnce, 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 AIPc and with HPD-PDT of T24 cells extracellular Ca 2+ 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., a20 2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.
Porphyrins in Tumor Phototherapy, 1984
Porphyrin photoradiation therapy for the treatment of various malignant tumors is a recent rapidl... more Porphyrin photoradiation therapy for the treatment of various malignant tumors is a recent rapidly developing technique and progress has been fast and promising1,2). The principle of porphyrin photoradiation therapy is relatively simple. Following systematic administration, porphyrins, especially the so-called hematoporphyrin-derivative (HpD) are accumulated to higher concentrations in malignant than in normal cells3). During subsequent exposure of the cells to visible light the accumulated porphyrin acts as photodynamic sensitizer 4) and causes oxidation of cellular constituent presumably by formation of singlet oxygen and possibly hydroxyl radicals. These oxidations lead to disturbed functions and, ultimately, cell death. Three mechanisms may be responsible e.g. membrane deterioration, DNA-damage and photodynamic inactivation of crucial enzyme systems. Studies of Kessel et al. indicate that membrane damage may be the direct cause of photodynamic cell death in tumors 5–7). Illumination of L1210 and SS-1 cells incubated with porphyrin leads to cross-linking of membrane proteins 6) as described before with red cell membranes 8,9). Further,loss of cell viability was correlated with inhibition of transmembrane cycloleucine transport 5).