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Prophylactic effects and spin trapping efficacies of 2-substituted DMPO spin traps
Session 6: Development of Antioxidant Pharmaceuticals 6:5
IMMUNOMODULATORY AND ANTIOXIDANT EFFECTS OF VITAMIN E ON HUMAN T-CELLS IN VITRO. Michael Osbandt, Jacob Shoham2, and Miriam Cohen*. ‘Odyssey Therapeutics, Inc., Newton Upper Falls, MA 02164 and *Department of Life Sciences, Bar Ban Univ, Ramat-Gan, Israel There is increasing awareness that vitamin E (a-tocopherol, Vit E) is clinically beneficial. The effects of Vit E on the immune system have been studied primarily in animals, or as part of studies on the dietary effects of Vit E on immune response. Very little is actually known about the effect of Vit E on human lymphocyte function. Consequently, we thought it would be interesting to assess the effect of Vit E on human T-cell function in vitro, with or without oxidative stress. Peripheral blood mononuclear cells (PBM) from healthy subjects of different ages (21-85 yrs) were prepared and suspended in media containing various concentrations of Vit E (.5 2 uM) and/or hydrogen peroxide (H&l*, 25-200 uM). The PBM were activated by the T-cell mitogens phytohemagglutinin or concanavalin A , and the proliferative response (PR) measured by 3H-thymidine incorporation after 3-8 days of culture. The results were: I) Vit E had a very strong effect, in a dose dependent manner, on PR of mitogen stimulated PBM, increasing the PR up to IO-fold; 2) The effect of Vit E was more pronounced in extended (6-8 day) than in short term (3 day) cultures; 3) Comparable effects were seen in both young and old subjects; 4) The greatest effect of Vit E was seen in the presence of strong oxidative stress. H,O, significantly reduced PR in a dose dependent fashion, with complete suppression of PR at the highest dose tested (200 uM). Vit E not only abolished this HP2 -induced suppression, and, in certain Hf12 doses (50 - 100 uM) actually caused the PR to overshoot to levels higher than with Vit E alone. These results indicate that Vit E has an exceptionally strong effect on T cells, and can salvage them from both endogenous and exogenous oxidative damage. Moreover, the extended proliferation assay described here can be used to monitor the biological effects of antioxidants on T-cells.
6:7
INVOLVEMENT OF FREE RADICALS IN IODGNIUM COMPOUND INHIBITION OF FLAVOENZYMES. V.B. O’Donnell, M.J. Burkitt, O.T.G. Jones*, Rowett Research Institute, Greenbum Road, Aberdeen, AB2 9SB, U.K. *Department of Biochemistry, University of Bristol, Bristol. BS8 ITD, U.K.
6:6
Reactive oxygen species have been implicated in the pathogenesis of many disease states. Therefore, the development of methods for control of their production has become a major pharmaceutical goal. In addition, as the diversity of effects of oxygen species as cellular signals become apparent, reliable tools allowing elucidation of oxygen radical-dependent events become essential. Diphenylene iodonium (DPI) and its analogues have been previously demonstrated as potent inhibitors of a range of flavoenzymes including neutrophil NADPH oxidase, nitric oxide synthase. NADH dehydrogenase, xanthine oxidase and cytochrome P450 reductase. Chemically, iodonium compounds are known as versatile arylating agents, mediating the addition of phenyl groups onto a wide variety of compounds. Reaction has been proposed to involve one-electron reduction of the inhibitor, leading to the formation of phenyl free radicals which then add back to the electron donor to form adducts. Previously, we have shown DPI to react with reduced, but not oxidised free flavin (FAD or FMN) forming phenylated adducts. In addition, inhibition of neutrophil NADPH oxidase was shown to have an absolute requirement for catalytic turnover and was associated with loss of native flavin from the enzyme. Recently, using e.s.r. techniques, we have obtained direct evidence for the generation of phenyl radicals following interaction of IDP with free flavins. and studies are underway to elucidate whether this mechanism in flavoenzyme also operates systems.
PROPHYLACTIC EFFECTS AND SPIN TRAPPING EFFICACIES OF 2-SUBSTITUTED DMPO SPIN TRAPS Yang-Kane Zhang, Larry D. Haire, J. Lee Poyer and Edward G. Janzen, National Biomedical Center for Spin Trapping and Free Radicals, Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA
MASS SPECTROMETRY OF HYDROXY-PBN’S Coit M. DuBose, Masana Arimura* and Edward G. Janzen National Biomedical Center for Spin Trapping and Free Radicals. Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA * on leave from the Department of Chemistry, Osaka University, Osaka, Japan
A number of 2-substituted DMPO spin traps have been synthesized. Testing has begun to determine the spin trapping efficiencies and prophylactic effects of these compounds. The use of the methyl group leads to unusually high artifact signal intensities by EPR but the choice of a 2-phenyl group improves this situation considerably. By labelling the nitronyl carbon with 13C the same kinds of triplets of doublets as found for PBN can be obtained from EPR spectra of spin adducts. Most spin adducts have long life-times because no E-hydrogen exists in the spin adduct for further degradative reactions. Spin trapping rate constants are being obtained to compare to PBN. In biological spin trapping the relative radical trapping efficiency is better for 2-phenyl DMPO than for PBN by about 2x; for example trichloromethyl from in vivo metabolism of CCI, in rat liver.
The mass spectra of substituted PBN’s have been obtained by electron impact. Of interest is the stabilization of the radical ion by electron-donating substituents. The parent ion in readily detected for amino, methoxy and hydroxy PBN’s. The spin trapping reaction between PBN and hydroxyl radicals produces the hydroxyl adduct which is unstable in aqueous solutions: Y. Kotake and E.G. Janzen, “Decay and Fate of Hydroxyl Radical Adduct of PBN in Aqueous Media”, J. Am. Chem. Sot. ll3,9503-9506 (1991). However other adducts must be formed wherein the phenyl ring is hydroxylatcd: P. Neta, S. Steenken, E.G. Janzen and R.V. Shetty, “Pattern of Addition of Hydroxyl Radicals to the Spin Trap 4-PyOBN,” J. Phys. Chem., 84, 532-534 (1980). This reaction is considered analogous to the hydroxylalion of salicylic acid and phenylalanine. Results from attempted detection of hydroxy PBN’s from systems producing hydroxyl radicals in the presence of PBN will be reported.
Other examples will be presented.
509
6:8
IMMUNOMODULATORY AND ANTIOXIDANT EFFECTS OF VITAMIN E ON HUMAN T-CELLS IN VITRO. Michael Osbandt, Jacob Shoham2, and Miriam Cohen*. ‘Odyssey Therapeutics, Inc., Newton Upper Falls, MA 02164 and *Department of Life Sciences, Bar Ban Univ, Ramat-Gan, Israel There is increasing awareness that vitamin E (a-tocopherol, Vit E) is clinically beneficial. The effects of Vit E on the immune system have been studied primarily in animals, or as part of studies on the dietary effects of Vit E on immune response. Very little is actually known about the effect of Vit E on human lymphocyte function. Consequently, we thought it would be interesting to assess the effect of Vit E on human T-cell function in vitro, with or without oxidative stress. Peripheral blood mononuclear cells (PBM) from healthy subjects of different ages (21-85 yrs) were prepared and suspended in media containing various concentrations of Vit E (.5 2 uM) and/or hydrogen peroxide (H&l*, 25-200 uM). The PBM were activated by the T-cell mitogens phytohemagglutinin or concanavalin A , and the proliferative response (PR) measured by 3H-thymidine incorporation after 3-8 days of culture. The results were: I) Vit E had a very strong effect, in a dose dependent manner, on PR of mitogen stimulated PBM, increasing the PR up to IO-fold; 2) The effect of Vit E was more pronounced in extended (6-8 day) than in short term (3 day) cultures; 3) Comparable effects were seen in both young and old subjects; 4) The greatest effect of Vit E was seen in the presence of strong oxidative stress. H,O, significantly reduced PR in a dose dependent fashion, with complete suppression of PR at the highest dose tested (200 uM). Vit E not only abolished this HP2 -induced suppression, and, in certain Hf12 doses (50 - 100 uM) actually caused the PR to overshoot to levels higher than with Vit E alone. These results indicate that Vit E has an exceptionally strong effect on T cells, and can salvage them from both endogenous and exogenous oxidative damage. Moreover, the extended proliferation assay described here can be used to monitor the biological effects of antioxidants on T-cells.
6:7
INVOLVEMENT OF FREE RADICALS IN IODGNIUM COMPOUND INHIBITION OF FLAVOENZYMES. V.B. O’Donnell, M.J. Burkitt, O.T.G. Jones*, Rowett Research Institute, Greenbum Road, Aberdeen, AB2 9SB, U.K. *Department of Biochemistry, University of Bristol, Bristol. BS8 ITD, U.K.
6:6
Reactive oxygen species have been implicated in the pathogenesis of many disease states. Therefore, the development of methods for control of their production has become a major pharmaceutical goal. In addition, as the diversity of effects of oxygen species as cellular signals become apparent, reliable tools allowing elucidation of oxygen radical-dependent events become essential. Diphenylene iodonium (DPI) and its analogues have been previously demonstrated as potent inhibitors of a range of flavoenzymes including neutrophil NADPH oxidase, nitric oxide synthase. NADH dehydrogenase, xanthine oxidase and cytochrome P450 reductase. Chemically, iodonium compounds are known as versatile arylating agents, mediating the addition of phenyl groups onto a wide variety of compounds. Reaction has been proposed to involve one-electron reduction of the inhibitor, leading to the formation of phenyl free radicals which then add back to the electron donor to form adducts. Previously, we have shown DPI to react with reduced, but not oxidised free flavin (FAD or FMN) forming phenylated adducts. In addition, inhibition of neutrophil NADPH oxidase was shown to have an absolute requirement for catalytic turnover and was associated with loss of native flavin from the enzyme. Recently, using e.s.r. techniques, we have obtained direct evidence for the generation of phenyl radicals following interaction of IDP with free flavins. and studies are underway to elucidate whether this mechanism in flavoenzyme also operates systems.
PROPHYLACTIC EFFECTS AND SPIN TRAPPING EFFICACIES OF 2-SUBSTITUTED DMPO SPIN TRAPS Yang-Kane Zhang, Larry D. Haire, J. Lee Poyer and Edward G. Janzen, National Biomedical Center for Spin Trapping and Free Radicals, Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA
MASS SPECTROMETRY OF HYDROXY-PBN’S Coit M. DuBose, Masana Arimura* and Edward G. Janzen National Biomedical Center for Spin Trapping and Free Radicals. Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 USA * on leave from the Department of Chemistry, Osaka University, Osaka, Japan
A number of 2-substituted DMPO spin traps have been synthesized. Testing has begun to determine the spin trapping efficiencies and prophylactic effects of these compounds. The use of the methyl group leads to unusually high artifact signal intensities by EPR but the choice of a 2-phenyl group improves this situation considerably. By labelling the nitronyl carbon with 13C the same kinds of triplets of doublets as found for PBN can be obtained from EPR spectra of spin adducts. Most spin adducts have long life-times because no E-hydrogen exists in the spin adduct for further degradative reactions. Spin trapping rate constants are being obtained to compare to PBN. In biological spin trapping the relative radical trapping efficiency is better for 2-phenyl DMPO than for PBN by about 2x; for example trichloromethyl from in vivo metabolism of CCI, in rat liver.
The mass spectra of substituted PBN’s have been obtained by electron impact. Of interest is the stabilization of the radical ion by electron-donating substituents. The parent ion in readily detected for amino, methoxy and hydroxy PBN’s. The spin trapping reaction between PBN and hydroxyl radicals produces the hydroxyl adduct which is unstable in aqueous solutions: Y. Kotake and E.G. Janzen, “Decay and Fate of Hydroxyl Radical Adduct of PBN in Aqueous Media”, J. Am. Chem. Sot. ll3,9503-9506 (1991). However other adducts must be formed wherein the phenyl ring is hydroxylatcd: P. Neta, S. Steenken, E.G. Janzen and R.V. Shetty, “Pattern of Addition of Hydroxyl Radicals to the Spin Trap 4-PyOBN,” J. Phys. Chem., 84, 532-534 (1980). This reaction is considered analogous to the hydroxylalion of salicylic acid and phenylalanine. Results from attempted detection of hydroxy PBN’s from systems producing hydroxyl radicals in the presence of PBN will be reported.
Other examples will be presented.
509
6:8