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Photosensitizing properties of tin and zinc porphyrin inhibitors of heme oxygenase
Photo-oxidative Stress and PhotodynamicTherapy
8.0
PHOTOOXIDATIVE STRESS IN THE EYE: ASCORBATE OXIDATION BY ACTINIC LIGHT Randolph D. Glickman and Kwok-Wai Lain. Dcp't. of Ophthalmology, University of Texas Health Science Center, San Antonio, TX 78284, USA. The production of free radicals during prolonged light exposure has been proposed as a mechanism for retinal degenerations. The oxidation of ascorbate, which is plentiful in ocular tissues, serves as a good indicator of photooxidative reactions. We have documented the oxidation of ascorbate into dehydroascorbate (DHA) in suspensions of retinal pigment epithelial (RPE) cells irradiated with the mixed, 488 + 514.5 nm output of an argon laser. RPE cells were brushed out of fresh bovine eyes and suspended in 0.15 M sucrose, 5 mM citrate, 0.005 mM EDTA, 0.17 mg/ml 14C-ascorbate (1 mCi), and placed in Amicon filter vials. The cells were then irradiated with laser light for 5 min at a power density of 127 mW/cm 2. The cells were separated from the incubation medium by centrifugation. Labelled DHA and ascorbate were separated by HPLC on a uBondapak column (Waters), and the isotope counted on a Radiomatic FLO-ONEq3eta. Analysis of the incubation media revealed that the DHA to ascorbate ratio was 2-3 times greater in the lasered samples than in the unexposed controls. The label inside the RPE cells eluted entirely in the DHA fraction. Oxidative stress induced by actinic light absorption evidently occurs at the plasma membranes of the RPE cells. Supported by grants from Alcon Labs (RDG), NEI EY08213 and The San Antonio Area Fdn (K-WL).
8.11
~ITIZING PROP~qFIES OF TIN AND ZINC P 0 ~ I~4IBITDRS OF H~4E OXYG~NASE. Nancy L. Greenbaum and Attallah Kappas, The Rockefeller University, New York, NY 10021 USA
77
8.10
Metalloporphyrin inhibitors of heine (the first and rate-limiting enzyme in the degradation of h~me to bilirubin), which have been shown to decrease serum bilirubin levels in vivo, may also have photosensitizing properties. To assess this potential in serum, the relative production of 102 mediated by Sn-proto- (SnPP), Sn-meso- (Sr~P), Sn-diiododeutero- (Sn~DP), and Zn-~rphyrin (z~MP) bound to h u ~ n serum albtmdn (HSA) was measured by the rate of 02 uptake associated with reaction of oxidizable amino acids of the protein plus added tryptophan. Illumination by continuous light in the Soret or visible regions of SnPP or SnMP bound to HSA in buffered solution resulted in a high rate of 02 uptake, but the rate mediated by Sn~DP was substantially less (3-9 times, dependiig upon tb~ buffer). The initial rate of 102 production sensitized by ZnMP bound to HSA was slightly greater than for the Sn analog. By ccm~oarison, the rate of 0 2 uptake mediated by metalloporphyrins dispersed in Triton X-100 (and therefore preduminantly in the monomer form, as when bound to HSA) was approximately the same for all ocmpourKis examined. While the Sn cumpounds bound to HSA demonstrated no significant destruction during the illtmdnation period, ZnMP was ~ y photolabile, and the resultant oxidation product was ineffective as an inhibitor of heme oxygenase.
SINGLET OXYGEN-MEDIATED GENERATION OF DYE-BASED TOXINS: IMPLICATIONS FOR SYSTEMIC THERAPY K.S. Gulliya, S. Pervaiz, T.C. Chanh, J. Newman, and J.L. Matthews. Baylor Research Foundation,Baylor University MedicalCenter, Dallas, TX 75246, U.S.A.
VITILIGOAND PHOTODYI~%MIC "I~U~KAPy Hoshnuwar Kerawalla, A.N.Inamdar, R.N.Shah. Department of Biochemistry, Institute of Science, Bombay - 400 032, India.
Memcyanine 540 (MC540) was preactivated by exposure to fluorescent light. The light-exposed (preactivated) P-MC540 was then used (in the dark) in cytotoxicity assays. Results show that P-MC540 was effective in killing several (but not all) types of tumor cells. The observed toxicity of P-MC540 to tumor ceils was significantly greater (p < 0.05) than the nonactivated MC540. The cell kill caused by P-MC540 was enhanced by buthionine sulfoxamine and decreased by eatalase and mannitol. A similar treatment of envelope viruses (HSV-1 and HIV-1) with P-MC540 resulted in virtually complete killing of viruses (ED50 and ED90, 10 ~tg/mL and 17.5 Ixg/mL, respectively). Similarly, syncytium formation, expression of core antigens, and reverse transcriptase activity was significantly reduced. P-MC540 retained 90% of its activity for five days at room temperature and >30 days at -135° C. The absorbance maxima of P-MC540 shifted to 280 nm indicating that the molecule is fragmented. The HPLC elution profiles of active and inactive P-MC540 were identical. However, the presenceof peroxideswas confirmedin P-MC540 and other preactivated photoactive dyes which correlated with biotoxicity. Addition of Fe(SO4)2 destroyed the biological activity by inactivationof peroxides. The actual oxidant may be H202 or radicals derived from it such as OH or HOCI. The involvement of other species is not ruled out and the mechanism of toxicity is not certain. P-MC540 was virtually nontoxic up to a dose of 200 mg/kg in mice and 20 mg/kg in dogs. The photoinduced electron transfer reactions seem not to be important. Extrapolation of our photophysical data on viscosity and diffusion length (10 nm/ns) for O2(IAg) indicate damage to the dye molecule and generation of dye-based toxins responsible for observed toxicity. The identity of the toxic specie will be discussed. Accordingly,preactivated photoactive compounds may have significant clinical applications. Supported by grants from the SDI MFEL Program, the Leukemia Association of North Central Texas, anti the Cell Biology Fund of the Baylor ResearchFoundation.
Vitiligo is a clinical condition where white patches appear on the skin due to loss of melanin. Melanin formation depends on the status of the enzyme, tyrosinase. It contains about 0.2% copper and is sythesised in the ribosomal fractions of the melanocytes. Psoralens have been used in the treatment of Vitiligo and are potent photodynamic agents. Orally given they act as photosensitive and photoprotective agents. 10-40 mg/day of 8-methoxypsoralen, divided into 2 doses, depending on the age of the patient was administered along with high-intensity UVA radiation (3 mins per week). Levels of copper, zinc, calcium and magnesium were measured before PUVA treatment by the inductively coupled plasma method in the sera of 42 patients and 20 normal individuals. Levels were: copper (0.224±0.087 ~g/ml), normal (0.8-1.5 ~g/ml); zinc (0.77±0.54~g/ml), normal (0.8-1.6~g/ml); calcium (3.93±2.53 Meq/l), normal (4.56.6 Meq/l); magnesium (2.22±2.04 Meq/l), normal (1.5-2.5 Meq/l). In a follow-up study, 15 cases were given zinc + PUVA treatment till complete pigmentation. 74% of these were found to have normal sera zinc levels. Results of the study favour the role of PUVA treatment supplemented with zinc therapy in the treatment of Vitiligo.
8.12
8.0
PHOTOOXIDATIVE STRESS IN THE EYE: ASCORBATE OXIDATION BY ACTINIC LIGHT Randolph D. Glickman and Kwok-Wai Lain. Dcp't. of Ophthalmology, University of Texas Health Science Center, San Antonio, TX 78284, USA. The production of free radicals during prolonged light exposure has been proposed as a mechanism for retinal degenerations. The oxidation of ascorbate, which is plentiful in ocular tissues, serves as a good indicator of photooxidative reactions. We have documented the oxidation of ascorbate into dehydroascorbate (DHA) in suspensions of retinal pigment epithelial (RPE) cells irradiated with the mixed, 488 + 514.5 nm output of an argon laser. RPE cells were brushed out of fresh bovine eyes and suspended in 0.15 M sucrose, 5 mM citrate, 0.005 mM EDTA, 0.17 mg/ml 14C-ascorbate (1 mCi), and placed in Amicon filter vials. The cells were then irradiated with laser light for 5 min at a power density of 127 mW/cm 2. The cells were separated from the incubation medium by centrifugation. Labelled DHA and ascorbate were separated by HPLC on a uBondapak column (Waters), and the isotope counted on a Radiomatic FLO-ONEq3eta. Analysis of the incubation media revealed that the DHA to ascorbate ratio was 2-3 times greater in the lasered samples than in the unexposed controls. The label inside the RPE cells eluted entirely in the DHA fraction. Oxidative stress induced by actinic light absorption evidently occurs at the plasma membranes of the RPE cells. Supported by grants from Alcon Labs (RDG), NEI EY08213 and The San Antonio Area Fdn (K-WL).
8.11
~ITIZING PROP~qFIES OF TIN AND ZINC P 0 ~ I~4IBITDRS OF H~4E OXYG~NASE. Nancy L. Greenbaum and Attallah Kappas, The Rockefeller University, New York, NY 10021 USA
77
8.10
Metalloporphyrin inhibitors of heine (the first and rate-limiting enzyme in the degradation of h~me to bilirubin), which have been shown to decrease serum bilirubin levels in vivo, may also have photosensitizing properties. To assess this potential in serum, the relative production of 102 mediated by Sn-proto- (SnPP), Sn-meso- (Sr~P), Sn-diiododeutero- (Sn~DP), and Zn-~rphyrin (z~MP) bound to h u ~ n serum albtmdn (HSA) was measured by the rate of 02 uptake associated with reaction of oxidizable amino acids of the protein plus added tryptophan. Illumination by continuous light in the Soret or visible regions of SnPP or SnMP bound to HSA in buffered solution resulted in a high rate of 02 uptake, but the rate mediated by Sn~DP was substantially less (3-9 times, dependiig upon tb~ buffer). The initial rate of 102 production sensitized by ZnMP bound to HSA was slightly greater than for the Sn analog. By ccm~oarison, the rate of 0 2 uptake mediated by metalloporphyrins dispersed in Triton X-100 (and therefore preduminantly in the monomer form, as when bound to HSA) was approximately the same for all ocmpourKis examined. While the Sn cumpounds bound to HSA demonstrated no significant destruction during the illtmdnation period, ZnMP was ~ y photolabile, and the resultant oxidation product was ineffective as an inhibitor of heme oxygenase.
SINGLET OXYGEN-MEDIATED GENERATION OF DYE-BASED TOXINS: IMPLICATIONS FOR SYSTEMIC THERAPY K.S. Gulliya, S. Pervaiz, T.C. Chanh, J. Newman, and J.L. Matthews. Baylor Research Foundation,Baylor University MedicalCenter, Dallas, TX 75246, U.S.A.
VITILIGOAND PHOTODYI~%MIC "I~U~KAPy Hoshnuwar Kerawalla, A.N.Inamdar, R.N.Shah. Department of Biochemistry, Institute of Science, Bombay - 400 032, India.
Memcyanine 540 (MC540) was preactivated by exposure to fluorescent light. The light-exposed (preactivated) P-MC540 was then used (in the dark) in cytotoxicity assays. Results show that P-MC540 was effective in killing several (but not all) types of tumor cells. The observed toxicity of P-MC540 to tumor ceils was significantly greater (p < 0.05) than the nonactivated MC540. The cell kill caused by P-MC540 was enhanced by buthionine sulfoxamine and decreased by eatalase and mannitol. A similar treatment of envelope viruses (HSV-1 and HIV-1) with P-MC540 resulted in virtually complete killing of viruses (ED50 and ED90, 10 ~tg/mL and 17.5 Ixg/mL, respectively). Similarly, syncytium formation, expression of core antigens, and reverse transcriptase activity was significantly reduced. P-MC540 retained 90% of its activity for five days at room temperature and >30 days at -135° C. The absorbance maxima of P-MC540 shifted to 280 nm indicating that the molecule is fragmented. The HPLC elution profiles of active and inactive P-MC540 were identical. However, the presenceof peroxideswas confirmedin P-MC540 and other preactivated photoactive dyes which correlated with biotoxicity. Addition of Fe(SO4)2 destroyed the biological activity by inactivationof peroxides. The actual oxidant may be H202 or radicals derived from it such as OH or HOCI. The involvement of other species is not ruled out and the mechanism of toxicity is not certain. P-MC540 was virtually nontoxic up to a dose of 200 mg/kg in mice and 20 mg/kg in dogs. The photoinduced electron transfer reactions seem not to be important. Extrapolation of our photophysical data on viscosity and diffusion length (10 nm/ns) for O2(IAg) indicate damage to the dye molecule and generation of dye-based toxins responsible for observed toxicity. The identity of the toxic specie will be discussed. Accordingly,preactivated photoactive compounds may have significant clinical applications. Supported by grants from the SDI MFEL Program, the Leukemia Association of North Central Texas, anti the Cell Biology Fund of the Baylor ResearchFoundation.
Vitiligo is a clinical condition where white patches appear on the skin due to loss of melanin. Melanin formation depends on the status of the enzyme, tyrosinase. It contains about 0.2% copper and is sythesised in the ribosomal fractions of the melanocytes. Psoralens have been used in the treatment of Vitiligo and are potent photodynamic agents. Orally given they act as photosensitive and photoprotective agents. 10-40 mg/day of 8-methoxypsoralen, divided into 2 doses, depending on the age of the patient was administered along with high-intensity UVA radiation (3 mins per week). Levels of copper, zinc, calcium and magnesium were measured before PUVA treatment by the inductively coupled plasma method in the sera of 42 patients and 20 normal individuals. Levels were: copper (0.224±0.087 ~g/ml), normal (0.8-1.5 ~g/ml); zinc (0.77±0.54~g/ml), normal (0.8-1.6~g/ml); calcium (3.93±2.53 Meq/l), normal (4.56.6 Meq/l); magnesium (2.22±2.04 Meq/l), normal (1.5-2.5 Meq/l). In a follow-up study, 15 cases were given zinc + PUVA treatment till complete pigmentation. 74% of these were found to have normal sera zinc levels. Results of the study favour the role of PUVA treatment supplemented with zinc therapy in the treatment of Vitiligo.
8.12