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Superoxide dismutase: An antioxidant in irradiated and non-irradiated food?
AntioxidantEnzymes 14.25
A N T I O X I D A N T E N Z Y M E L E V E L S IN N O R M A L AND CYTOKINE-PRIMED HUMAN N E U T R O P H I L S . Michael L. McCormick, Larry W. Oberley and Bradley E. Britigan. V A Medical Ctr. Research Service, Dept. of Medicine and Radiation Research Lab, Univ. of Iowa, Iowa City, I A 52242.
Neutrophils (PMNs) function in vivo in the presence of highly damaging reactive oxygen intermediates (ROI), yet little is k n o w n about their antioxidant mechanisms. Western immunoblot analysis after subcellular fractionation of N2-cavitated PMNs on a discontinuous Percoll gradient showed > 95% of the MnSOD immunoreactivity to be associated with the plasma membrane. This was in contrast to CuZnSOD which was seen only in cytosol and catalase of which - 25% was in the plasma m e m b r a n e and 75% in cytosol. No antioxidant enzyme immunoreactivity was seen in either primary or specific granules. PMNs are nearly devoid of mitochondria. A comparison of immunoreactive protein and enzymatic activity showed good correlation for CuZnSOD and catalase in PMNs. However, normal resting PMNs contained considerable MnSOD immunoreactivity that was not accounted for by enzymatic activity. Cytokines such as TNF-a and IL-1B, prime PMNs for enhanced production of 0 2- in response to P M A and other stimuli. Treatment of PMNs with priming doses of TNF-a and IL-1B did not increase immunoreactive levels of MnSOD, CuZnSOD, or catalase either by immunoblot analysis or activity gel. These data do not support a link between cytokine priming of P M N O~" production and induction of antioxidant enyzmes. e novel possibility of MnSOD localizing to plasma m e m b r a n e could be important in PMN membrane protection as well as modulation of toxic ROI produced following N A D P H oxidase activation.
14.27
EVIDENCE THAT RAT LIVER MICROSOMAL GLUTATHIONE TRANSFERASE IS RESPONSIBLE FOR GLUTATHIONE-DEPENDENT PROTECTION AGAINST LIPID PEROXIDATION. Eriflli Mosialou, Anton E. P. Adang1 and Rail Morgenstem. Department of Toxicology, Karolinska Instituter, Box 60400, S-10401 Stockholm, Sweden and 1the Department of Organic Chemistry, University of Leiden, Leiden, The Netherlands.
133
14.26
Evidence that rat liver microsomal glutathione transferase is involved in glutathione-dependent inhibition of lipid peroxidation in the liver has been obtained. Activation of the enzyme in microsomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared to untreated microsomes. Earlier findings that N-ethylmaleimide abolishes glutathionedependent protection against lipid peroxidation, which were taken as evidence against the involvment of microsomal glutathione transferase, were found to depend critically on the experimental system used. Our findings, in contrast, show that N-ethylmaleimide does not remove the protective effect of glutathione (5 mM). Upon examining the effect of seven glutathione analogues on lipid peroxidation it was found that only those that serve as good substrates for the microsomal glutathione transferase can inhibit lipid peroxidation. The total lack of inhibition by for example the 7" D-Glu-L-Cys-Gly analogue shows the specificity of lhe protection and rules out any non-enzymic component. Finally an inhibitor of selenium-dependent glutathione peroxidase (meroaptosuccinate at 501~M)does not inhibit the glutathione-dependent protection of rat liver microsomes against lipid peroxidation. In conclusion, the evidence favors the involvment of microsomal glutathione transferase in the protection against lipid peroxidation. The protection is propably a consequence of the ability of the enzyme to function as a glutatione peroxidase towards lipid hydroperoxides. Supported by the Swedish Cancer Society.
EVIDENCE FOR A HYDROPEROXIDE REDUCTASE IN PLASMA Paul A. Motchnik, Balz Frei and Bruce N. Ames Division of Biochem. and Molecular Biol. The University of California, Berkeley, CA 94720
SUPEROXIDE DISMUTASE: AN ANTIOXIDANT IN IRRADIATED AND NON-IRRADIATED FOOD? Deborah J. Nice, Judith K. Donnelly and David S. Robinson. Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK.
Oxidation of lipids in LDL has been proposed to be involved in athemgenesis. The breakdown of lipid hydroperoxides in plasma may be important in this and other diseases associated with oxidative stress. Haman and rat plasma are capable of degrading exogenously added free fatty acid hydroperoxides and cumene hydroperoxide (CuOOH). We have been studying the degradation of CuOOH in blood plasma from the rat. CuOOH added to plasma was quantitatively reduced to cumyi alcohol. Protease-treatment of plasma resulted in a decrease in CuOOH degradation. CuOOH degradation was not affected by inhibitors of selenium-dependent glutathione peroxidase, glutathione transfemse, or lipoxygenase, enzymes known to reduce hydroperoxides. Dietary selenium deficiency did not affect CuOOH degradation, although glutathione peroxidase activity was decreased 75%. When plasma was passed through a Sephadex G-25 column the flowthrough, or high molecular weight fraction (HMW), had less reductase activity than whole plasma. However a mixture of HMW + plasma had more reductase activity than BSA + plasma. This suggests that the HMW fraction contains the hydroperoxide reductase but the enzyme requires a low molecular weight component in whole plasma for activity. The identity of the reducing cofactor for the plasma hydroperoxide reductase is currently under investigation.
Studies were carried out on aqueous emulsified linoleic acid (2.5-10rn.M) model system. Addition of bovine erythrocyte superoxide dismutase (BSOD, 0.5-101.tg/ml) prolonged the lag period of the autoxidation process as indicated by the formation of the conjugated diene interraediate product, whereas addition of either heroin (10-15gM) or heat inactivated BSOD reduced the length of the lag phaseconsiderably and it was totally abolishedby addition of 100p.M heroin. BSOD appearsto retard autoxidation by prolonging the initiation step (lag period). BSOD also retards the heroin cata/ysedoxidation of linoleic acid, but by lowering the maximum rate of diene formation during the propagation period and not affecting the length of the lag period. Gamma irradiationinduced linoleic acid oxidation was also investigated. Again the SOD retarded oxidation upon storage but it had little affect upon diene formation during the irradiation period. Following an irradiation doseof 0.5 KGy, ie. that capableof delaying ripening in fruits and sprouting in vegetables, BSOD (ll~g/ml) retarded oxidation of linoleic acid considerably and BSOD concentrations as low as 0,05 Izg/ml showed some retardation, Comparisonswith the synthetic chain breaking antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluone (BHT) at 301.tg/ml, showed that retardation of oxidation was considerably less with BSOD. However consumer demands may in future limit the use of these synthetic compounds in foods, in favour of naturally occurring antioxidants such as SOD.
14.28
A N T I O X I D A N T E N Z Y M E L E V E L S IN N O R M A L AND CYTOKINE-PRIMED HUMAN N E U T R O P H I L S . Michael L. McCormick, Larry W. Oberley and Bradley E. Britigan. V A Medical Ctr. Research Service, Dept. of Medicine and Radiation Research Lab, Univ. of Iowa, Iowa City, I A 52242.
Neutrophils (PMNs) function in vivo in the presence of highly damaging reactive oxygen intermediates (ROI), yet little is k n o w n about their antioxidant mechanisms. Western immunoblot analysis after subcellular fractionation of N2-cavitated PMNs on a discontinuous Percoll gradient showed > 95% of the MnSOD immunoreactivity to be associated with the plasma membrane. This was in contrast to CuZnSOD which was seen only in cytosol and catalase of which - 25% was in the plasma m e m b r a n e and 75% in cytosol. No antioxidant enzyme immunoreactivity was seen in either primary or specific granules. PMNs are nearly devoid of mitochondria. A comparison of immunoreactive protein and enzymatic activity showed good correlation for CuZnSOD and catalase in PMNs. However, normal resting PMNs contained considerable MnSOD immunoreactivity that was not accounted for by enzymatic activity. Cytokines such as TNF-a and IL-1B, prime PMNs for enhanced production of 0 2- in response to P M A and other stimuli. Treatment of PMNs with priming doses of TNF-a and IL-1B did not increase immunoreactive levels of MnSOD, CuZnSOD, or catalase either by immunoblot analysis or activity gel. These data do not support a link between cytokine priming of P M N O~" production and induction of antioxidant enyzmes. e novel possibility of MnSOD localizing to plasma m e m b r a n e could be important in PMN membrane protection as well as modulation of toxic ROI produced following N A D P H oxidase activation.
14.27
EVIDENCE THAT RAT LIVER MICROSOMAL GLUTATHIONE TRANSFERASE IS RESPONSIBLE FOR GLUTATHIONE-DEPENDENT PROTECTION AGAINST LIPID PEROXIDATION. Eriflli Mosialou, Anton E. P. Adang1 and Rail Morgenstem. Department of Toxicology, Karolinska Instituter, Box 60400, S-10401 Stockholm, Sweden and 1the Department of Organic Chemistry, University of Leiden, Leiden, The Netherlands.
133
14.26
Evidence that rat liver microsomal glutathione transferase is involved in glutathione-dependent inhibition of lipid peroxidation in the liver has been obtained. Activation of the enzyme in microsomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared to untreated microsomes. Earlier findings that N-ethylmaleimide abolishes glutathionedependent protection against lipid peroxidation, which were taken as evidence against the involvment of microsomal glutathione transferase, were found to depend critically on the experimental system used. Our findings, in contrast, show that N-ethylmaleimide does not remove the protective effect of glutathione (5 mM). Upon examining the effect of seven glutathione analogues on lipid peroxidation it was found that only those that serve as good substrates for the microsomal glutathione transferase can inhibit lipid peroxidation. The total lack of inhibition by for example the 7" D-Glu-L-Cys-Gly analogue shows the specificity of lhe protection and rules out any non-enzymic component. Finally an inhibitor of selenium-dependent glutathione peroxidase (meroaptosuccinate at 501~M)does not inhibit the glutathione-dependent protection of rat liver microsomes against lipid peroxidation. In conclusion, the evidence favors the involvment of microsomal glutathione transferase in the protection against lipid peroxidation. The protection is propably a consequence of the ability of the enzyme to function as a glutatione peroxidase towards lipid hydroperoxides. Supported by the Swedish Cancer Society.
EVIDENCE FOR A HYDROPEROXIDE REDUCTASE IN PLASMA Paul A. Motchnik, Balz Frei and Bruce N. Ames Division of Biochem. and Molecular Biol. The University of California, Berkeley, CA 94720
SUPEROXIDE DISMUTASE: AN ANTIOXIDANT IN IRRADIATED AND NON-IRRADIATED FOOD? Deborah J. Nice, Judith K. Donnelly and David S. Robinson. Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK.
Oxidation of lipids in LDL has been proposed to be involved in athemgenesis. The breakdown of lipid hydroperoxides in plasma may be important in this and other diseases associated with oxidative stress. Haman and rat plasma are capable of degrading exogenously added free fatty acid hydroperoxides and cumene hydroperoxide (CuOOH). We have been studying the degradation of CuOOH in blood plasma from the rat. CuOOH added to plasma was quantitatively reduced to cumyi alcohol. Protease-treatment of plasma resulted in a decrease in CuOOH degradation. CuOOH degradation was not affected by inhibitors of selenium-dependent glutathione peroxidase, glutathione transfemse, or lipoxygenase, enzymes known to reduce hydroperoxides. Dietary selenium deficiency did not affect CuOOH degradation, although glutathione peroxidase activity was decreased 75%. When plasma was passed through a Sephadex G-25 column the flowthrough, or high molecular weight fraction (HMW), had less reductase activity than whole plasma. However a mixture of HMW + plasma had more reductase activity than BSA + plasma. This suggests that the HMW fraction contains the hydroperoxide reductase but the enzyme requires a low molecular weight component in whole plasma for activity. The identity of the reducing cofactor for the plasma hydroperoxide reductase is currently under investigation.
Studies were carried out on aqueous emulsified linoleic acid (2.5-10rn.M) model system. Addition of bovine erythrocyte superoxide dismutase (BSOD, 0.5-101.tg/ml) prolonged the lag period of the autoxidation process as indicated by the formation of the conjugated diene interraediate product, whereas addition of either heroin (10-15gM) or heat inactivated BSOD reduced the length of the lag phaseconsiderably and it was totally abolishedby addition of 100p.M heroin. BSOD appearsto retard autoxidation by prolonging the initiation step (lag period). BSOD also retards the heroin cata/ysedoxidation of linoleic acid, but by lowering the maximum rate of diene formation during the propagation period and not affecting the length of the lag period. Gamma irradiationinduced linoleic acid oxidation was also investigated. Again the SOD retarded oxidation upon storage but it had little affect upon diene formation during the irradiation period. Following an irradiation doseof 0.5 KGy, ie. that capableof delaying ripening in fruits and sprouting in vegetables, BSOD (ll~g/ml) retarded oxidation of linoleic acid considerably and BSOD concentrations as low as 0,05 Izg/ml showed some retardation, Comparisonswith the synthetic chain breaking antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluone (BHT) at 301.tg/ml, showed that retardation of oxidation was considerably less with BSOD. However consumer demands may in future limit the use of these synthetic compounds in foods, in favour of naturally occurring antioxidants such as SOD.
14.28