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Protection against free radical injury by selenoenzymes
Pharmac. Ther. Vol. 45, pp. 383-385, 1990 Printed in Great Britain. All rights reserved
0163-7258/90 $0.00 + 0.50 © 1989 Pergamon Press plc
Specialist Subject Editor: J. L. HOLTZMAN
P R O T E C T I O N AGAINST FREE RADICAL INJURY BY SELENOENZYMES RAYMONDF. BURK Division of Gastroenterology, Department of Medicine and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, U.S.A.
In 1957 selenium was demonstrated to protect rats against dietary liver necrosis (Schwarz and Foltz, 1957). Because the condition could also be prevented by the free radical-scavenger vitamin E, it was postulated that selenium might function by protecting against free radical injury. Years later dietary liver necrosis was shown to be accompanied by lipid peroxidation, and this free radical-mediated process could be prevented by adding selenium to a vitamin E- and selenium-deficient diet (Hafeman and Hoekstra, 1977). This strengthened the hypothesis that selenium serves in vivo as an oxidant defense. Selenium exerts its biological activity as a constituent of selenoproteins. To date three selenoproteins have been purified from animals and evidence has been presented that several more exist (Behne et aL, 1988). A complete understanding of the antioxidant effect of selenium has not been achieved. However, each of the three selenoproteins has been proposed to serve in oxidant defense. We will consider them in turn. GLUTATHIONEPEROXIDASE In 1973 glutathione peroxidase was found to contain selenium (Rotruck et al., 1973). Deficiency of the element caused a fall in the activity of this enzyme to very low levels, often to less than 5% of control activity. This discovery provided a basis for the antioxidant effect of selenium. It was envisioned that vitamin E scavenged free radicals, thus preventing hydroperoxide formation. If hydroperoxides formed, however, glutathione peroxidase could metabolize them, preventing their breakdown to free radical products which could perpetuate lipid peroxidation (Hoekstra, 1975). Selenium-dependent glutathione peroxidase can metabolize hydrogen peroxide to water. It and catalase are the enzymes which control hydrogen peroxide concentrations in cells (Hoekstra, 1975; Oshino et al., 1975). Hydrogen peroxide can react to yield free radicals and impair numerous processes in the cell. Hydrogen peroxide concentrations have not been measured in selenium-deficient cells but they would be expected to be elevated. Such an elevation of hydrogen peroxide concentration might lead to oxidant injury under certain conditions. Several laboratories have studied glutathione-dependent protection against lipid peroxidation in liver microsomal systems. An early report suggested that glutathione peroxidase prevented lipid peroxidation (McCay et al., 1976) but later work showed that this protection was due to other proteins and was not related to the selenium-dependent glutathione peroxidase (Burk et al., 1980b). Several groups attacked this problem directly and showed that selenium-dependent glutathione peroxidase cannot utilize as substrates fatty acid hydroperoxides which are esterified in phospholipids (Grossmann and Wendel, 1983). The activity of phospholipase A 2 is needed to provide free fatty acid hydroperoxide which can be metabolized by glutathione peroxidase (Grossman and Wendel, 1983; Sevanian et al., 1983). A formal comparison of selenium-dependent glutathione peroxidase with other glutathione-dependent factors has not been carried out, but scrutiny of existing 383
384
R.F. BURK
studies suggests that it provides relatively poor protection (McCay et al., 1976; Burk et al., 1980b). Thus, there is only weak evidence on a biochemical level that seleniumdependent glutathione peroxidase functions as an oxidant defense. Evidence has also been sought in in vivo studies that selenium-dependent glutathione peroxidase is an oxidant defense. Selenium-deficient rats given relatively small doses of diquat die within several hours, after developing high rates of lipid peroxidation and massive liver necrosis (Burk et al., 1980a). Administration of a physiological dose of selenium 10 hours before the diquat provided strong protection against the lipid peroxidation and the liver necrosis and improved survival. However, glutathione peroxidase activity in liver, kidney, and plasma responded only minimally (Burk et al., 1980b). Thus the protection by selenium could not be correlated with a rise in glutathione peroxidase activity. Another group studied paraquat lethality in chickens and had similar findings (Mercurio and Combs, 1986). While supporting an oxidant defense role for selenium, these reports suggest it might be exerted through a function other than glutathione peroxidase. The physiological role of selenium-dependent glutathione peroxidase is not known. Although its activity would appear to qualify it as an oxidant defense, evidence supporting such a function has not been presented. A major fraction of body selenium is in the form of glutathione peroxidase, however (Behne and Wolters, 1983), and the activity of this enzyme readily falls when dietary selenium intake is restricted. It is possible that glutathione peroxidase serves as a storage form of selenium and that it can release its selenium for use in other selenoproteins when the element is in short supply. PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE
While pursuing the glutathione-dependent activity in liver cytosol which prevents microsomal lipid peroxidation, Ursini and coworkers isolated a selenium-containing protein which has a glutathione peroxidase activity different from the classic selenium-dependent glutathione peroxidase (Ursini et al., 1982). This enzyme has been designated phospholipid hydroperoxide glutathione peroxidase because it can metabolize fatty acid hydroperoxides which are esterified in phospholipids. Another group has reported isolating a similar activity from rat liver but did not indicate whether it contained selenium (Duan et al., 1988). Addition of phospholipid hydroperoxide glutathione peroxidase to microsomal lipid peroxidation systems blocks lipid peroxidation (Ursini et al., 1982). Thus, this enzyme might well constitute an oxidant defense. So far assays of this activity have not been reported in selenium deficiency and routine assays are not possible. A great deal of work is needed to assess the role of this selenoprotein in protecting against free radicals. SELENOPROTEINP Several reports in the 1970's suggested that there was a plasma selenoprotein distinct from glutathione peroxidase (Herrman, 1977). In 1987 this protein was purified by immunoaffinity chromatography and a radioimmunoassay for it was devised (Yang et al., 1987). Selenoprotein P is a glycoprotein, containing selenium as selenocysteine (Motsenbocker and Tappel, 1982). It is made in the liver and apparently secreted into the plasma. Selenium deficiency in the rat depresses the level of selenoprotein P to less than 10% of control (Yang et al., 1987). Feeding intermediate dietary levels of selenium lowers selenoprotein P concentrations, but the decreases are somewhat less than the decreases in glutathione peroxidase activity (Yang et al., 1989). In addition, administration of selenium to a selenium-deficient rat leads to a rapid increase in plasma selenoprotein P levels. Twelve hours after injection of 50 #g of selenium, the concentration was 78% of control (Hill et al., 1988). Glutathione peroxidase activity remained less than 5% of control. One group has speculated that selenoprotein P is a selenium transport protein (Motsenbocker and Tappel, 1982), based on its rapid synthesis in the liver and plasma location. They have provided evidence for receptors for the protein located in the testis (Motchnik et al., 1987). Our group has suggested that selenoprotein P might be an oxidant
Protection against free radical injury by selenoenzymes
385
defense protein (Yang et al., 1987). This is based on its appearance in the time frame of selenium protection against diquat injury (see above). Further work will be necessary to determine the function(s) of selenoprotein P. CONCLUSIONS
It seems clear, based on its protective effect against dietary liver necrosis and diquat-induced lipid peroxidation, that selenium plays an important role in protecting against free-radical injury. Definition of that role at a metabolic level has proved difficult, however. While glutathione peroxidase is an attractive candidate for the oxidant defense form of selenium, most studies have not been able to implicate it as an oxidant defense. The two recently-isolated selenoproteins are presently being evaluated for oxidant defense properties. The protective effect of selenium against free radicals may reside in them or in selenoproteins which have not yet been characterized. REFERENCES BEnNE, D. and WOLrERS,W. (1983) Distribution of selenium and glutathione peroxidase in the rat. J. Nutr. 113: 456-461. BEHNE, D., HILMERT,H., SCnEID, S., GESSNER,H. and ELOER,W. (1988) Evidence for specific selenium target tissues and new biologically important selenoproteins. Biochim. Biophys. Acta 966: 1251. B~RK, R. F., LAWRENCE,R. A. and LANE,J. M. (1980a) Liver necrosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. J. Clin. Invest. 6S: 1024-1031. BURK, R. F., TRUMBLE,M. J. and LAWRENCE,R. A. (1980b) Rat hepatic cytosolic glutathione-dependent enzyme protection against lipid peroxidation in the NADPH-microsomal lipid peroxidation system. Biochim. Biophys. Acta 618: 35-41. DUAN,Y.-J., KOMURA,S., FISZER-SZAFARZ,B., SZAFARZ,D. and YAGI,K. (1988) Purification and characterization of a novel monomeric glutathione peroxidase from rat liver. J. Biol. Chem. 263: 19003-19008. GROSSMANN, A. and WENDEL, A. (1983) Non-reactivity of the selenoenzyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids. Cur. J. Biochem. 135: 549-552. HaFEMAN,D. G. and HOEKSTRA,W. G. (1977) Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J. Nutr. 107: 666-672. HERRMAN,J. L. (1977) The properties of a rat serum protein labelled by the injection of sodium selenite. Biochim. Biophys. Acta 500:61 70. HILL, K. E., YANG, J.-G. and BtSRK, R. F. (1988) Assessment of selenium intake: comparison of selenoprotein P concentration and glutathione peroxidase activity in rat plasma. F A S E B J. 2: A1088. HOEKSTRA,W. G. (1975) Biochemical function of selenium and its relation to vitamin E. Fed. Proc. 34: 2083-2089. MCCAV, P. B., GIBSON,D. D., FONG,K.-L. and HORNBROOK,K. R. (1976) Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochim. Biophys. Acta 431: 459-468. MERCURIO,S. D. and COMBS,G. F. (1986) Selenium-dependent glutathione peroxidase inhibitors increase toxicity of pro-oxidant compounds. J. Nutr. 116: 1726-1734. MOTCHNIK,P. A., GOMEZ,B. and TAPPEL,A. L. (1987) Rat plasma selenoprotein characterization and membrane receptor binding properties. Fed. Proc. 46: 907A. MOTSENBOCKER,M. A. and TAPPEL,A. L. (1982) A selenocysteine-containing selenium-transport protein in rat plasma. Biochim. Biophys. Acta 719: 147-153. OsmNo, N., JAMIESON,D. and CHANCE, B. (1975) The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver. Biochem. J. 146: 53~5. ROTRt;CK, J. T., POPE, A. L., GANTHER,H. E., SWANSON,A. B., HA~MAN, D. G. and HOEKSTRA,W. G. (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179: 585-590. SCHWARZ, K. and FOLTZ, C. M. (1957) Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: 3292-3293. SEVAN1AN,A., MUAKKASSAH-KELLV,S. F. and MONTESTRt;QUE,S. (1983) The influence of phospholipase A 2 and glutathione peroxidase on the elimination of membrane lipid peroxides. Arch. Biochem. Biophys. 223: 441-452. URSINt, F., MA1ORINO,M., VALENTE,M., FERRh L. and GREGOUN,C. (1982) Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochim. Biophys. Acta 710: 197-211. YANG, J.-G., MORRISON-PLUMrCmR,J. and Bt;RK, R. F. (1987) Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies. J. Biol. Chem. 262: 13372-13375. YANG, J.-G., HILt, K. E. and BURK, R. F. (1989) Dietary selenium intake controls rat plasma selenoprotein P concentration. J. Nutr. 119: 1010-1012.
0163-7258/90 $0.00 + 0.50 © 1989 Pergamon Press plc
Specialist Subject Editor: J. L. HOLTZMAN
P R O T E C T I O N AGAINST FREE RADICAL INJURY BY SELENOENZYMES RAYMONDF. BURK Division of Gastroenterology, Department of Medicine and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, U.S.A.
In 1957 selenium was demonstrated to protect rats against dietary liver necrosis (Schwarz and Foltz, 1957). Because the condition could also be prevented by the free radical-scavenger vitamin E, it was postulated that selenium might function by protecting against free radical injury. Years later dietary liver necrosis was shown to be accompanied by lipid peroxidation, and this free radical-mediated process could be prevented by adding selenium to a vitamin E- and selenium-deficient diet (Hafeman and Hoekstra, 1977). This strengthened the hypothesis that selenium serves in vivo as an oxidant defense. Selenium exerts its biological activity as a constituent of selenoproteins. To date three selenoproteins have been purified from animals and evidence has been presented that several more exist (Behne et aL, 1988). A complete understanding of the antioxidant effect of selenium has not been achieved. However, each of the three selenoproteins has been proposed to serve in oxidant defense. We will consider them in turn. GLUTATHIONEPEROXIDASE In 1973 glutathione peroxidase was found to contain selenium (Rotruck et al., 1973). Deficiency of the element caused a fall in the activity of this enzyme to very low levels, often to less than 5% of control activity. This discovery provided a basis for the antioxidant effect of selenium. It was envisioned that vitamin E scavenged free radicals, thus preventing hydroperoxide formation. If hydroperoxides formed, however, glutathione peroxidase could metabolize them, preventing their breakdown to free radical products which could perpetuate lipid peroxidation (Hoekstra, 1975). Selenium-dependent glutathione peroxidase can metabolize hydrogen peroxide to water. It and catalase are the enzymes which control hydrogen peroxide concentrations in cells (Hoekstra, 1975; Oshino et al., 1975). Hydrogen peroxide can react to yield free radicals and impair numerous processes in the cell. Hydrogen peroxide concentrations have not been measured in selenium-deficient cells but they would be expected to be elevated. Such an elevation of hydrogen peroxide concentration might lead to oxidant injury under certain conditions. Several laboratories have studied glutathione-dependent protection against lipid peroxidation in liver microsomal systems. An early report suggested that glutathione peroxidase prevented lipid peroxidation (McCay et al., 1976) but later work showed that this protection was due to other proteins and was not related to the selenium-dependent glutathione peroxidase (Burk et al., 1980b). Several groups attacked this problem directly and showed that selenium-dependent glutathione peroxidase cannot utilize as substrates fatty acid hydroperoxides which are esterified in phospholipids (Grossmann and Wendel, 1983). The activity of phospholipase A 2 is needed to provide free fatty acid hydroperoxide which can be metabolized by glutathione peroxidase (Grossman and Wendel, 1983; Sevanian et al., 1983). A formal comparison of selenium-dependent glutathione peroxidase with other glutathione-dependent factors has not been carried out, but scrutiny of existing 383
384
R.F. BURK
studies suggests that it provides relatively poor protection (McCay et al., 1976; Burk et al., 1980b). Thus, there is only weak evidence on a biochemical level that seleniumdependent glutathione peroxidase functions as an oxidant defense. Evidence has also been sought in in vivo studies that selenium-dependent glutathione peroxidase is an oxidant defense. Selenium-deficient rats given relatively small doses of diquat die within several hours, after developing high rates of lipid peroxidation and massive liver necrosis (Burk et al., 1980a). Administration of a physiological dose of selenium 10 hours before the diquat provided strong protection against the lipid peroxidation and the liver necrosis and improved survival. However, glutathione peroxidase activity in liver, kidney, and plasma responded only minimally (Burk et al., 1980b). Thus the protection by selenium could not be correlated with a rise in glutathione peroxidase activity. Another group studied paraquat lethality in chickens and had similar findings (Mercurio and Combs, 1986). While supporting an oxidant defense role for selenium, these reports suggest it might be exerted through a function other than glutathione peroxidase. The physiological role of selenium-dependent glutathione peroxidase is not known. Although its activity would appear to qualify it as an oxidant defense, evidence supporting such a function has not been presented. A major fraction of body selenium is in the form of glutathione peroxidase, however (Behne and Wolters, 1983), and the activity of this enzyme readily falls when dietary selenium intake is restricted. It is possible that glutathione peroxidase serves as a storage form of selenium and that it can release its selenium for use in other selenoproteins when the element is in short supply. PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE
While pursuing the glutathione-dependent activity in liver cytosol which prevents microsomal lipid peroxidation, Ursini and coworkers isolated a selenium-containing protein which has a glutathione peroxidase activity different from the classic selenium-dependent glutathione peroxidase (Ursini et al., 1982). This enzyme has been designated phospholipid hydroperoxide glutathione peroxidase because it can metabolize fatty acid hydroperoxides which are esterified in phospholipids. Another group has reported isolating a similar activity from rat liver but did not indicate whether it contained selenium (Duan et al., 1988). Addition of phospholipid hydroperoxide glutathione peroxidase to microsomal lipid peroxidation systems blocks lipid peroxidation (Ursini et al., 1982). Thus, this enzyme might well constitute an oxidant defense. So far assays of this activity have not been reported in selenium deficiency and routine assays are not possible. A great deal of work is needed to assess the role of this selenoprotein in protecting against free radicals. SELENOPROTEINP Several reports in the 1970's suggested that there was a plasma selenoprotein distinct from glutathione peroxidase (Herrman, 1977). In 1987 this protein was purified by immunoaffinity chromatography and a radioimmunoassay for it was devised (Yang et al., 1987). Selenoprotein P is a glycoprotein, containing selenium as selenocysteine (Motsenbocker and Tappel, 1982). It is made in the liver and apparently secreted into the plasma. Selenium deficiency in the rat depresses the level of selenoprotein P to less than 10% of control (Yang et al., 1987). Feeding intermediate dietary levels of selenium lowers selenoprotein P concentrations, but the decreases are somewhat less than the decreases in glutathione peroxidase activity (Yang et al., 1989). In addition, administration of selenium to a selenium-deficient rat leads to a rapid increase in plasma selenoprotein P levels. Twelve hours after injection of 50 #g of selenium, the concentration was 78% of control (Hill et al., 1988). Glutathione peroxidase activity remained less than 5% of control. One group has speculated that selenoprotein P is a selenium transport protein (Motsenbocker and Tappel, 1982), based on its rapid synthesis in the liver and plasma location. They have provided evidence for receptors for the protein located in the testis (Motchnik et al., 1987). Our group has suggested that selenoprotein P might be an oxidant
Protection against free radical injury by selenoenzymes
385
defense protein (Yang et al., 1987). This is based on its appearance in the time frame of selenium protection against diquat injury (see above). Further work will be necessary to determine the function(s) of selenoprotein P. CONCLUSIONS
It seems clear, based on its protective effect against dietary liver necrosis and diquat-induced lipid peroxidation, that selenium plays an important role in protecting against free-radical injury. Definition of that role at a metabolic level has proved difficult, however. While glutathione peroxidase is an attractive candidate for the oxidant defense form of selenium, most studies have not been able to implicate it as an oxidant defense. The two recently-isolated selenoproteins are presently being evaluated for oxidant defense properties. The protective effect of selenium against free radicals may reside in them or in selenoproteins which have not yet been characterized. REFERENCES BEnNE, D. and WOLrERS,W. (1983) Distribution of selenium and glutathione peroxidase in the rat. J. Nutr. 113: 456-461. BEHNE, D., HILMERT,H., SCnEID, S., GESSNER,H. and ELOER,W. (1988) Evidence for specific selenium target tissues and new biologically important selenoproteins. Biochim. Biophys. Acta 966: 1251. B~RK, R. F., LAWRENCE,R. A. and LANE,J. M. (1980a) Liver necrosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. J. Clin. Invest. 6S: 1024-1031. BURK, R. F., TRUMBLE,M. J. and LAWRENCE,R. A. (1980b) Rat hepatic cytosolic glutathione-dependent enzyme protection against lipid peroxidation in the NADPH-microsomal lipid peroxidation system. Biochim. Biophys. Acta 618: 35-41. DUAN,Y.-J., KOMURA,S., FISZER-SZAFARZ,B., SZAFARZ,D. and YAGI,K. (1988) Purification and characterization of a novel monomeric glutathione peroxidase from rat liver. J. Biol. Chem. 263: 19003-19008. GROSSMANN, A. and WENDEL, A. (1983) Non-reactivity of the selenoenzyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids. Cur. J. Biochem. 135: 549-552. HaFEMAN,D. G. and HOEKSTRA,W. G. (1977) Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution. J. Nutr. 107: 666-672. HERRMAN,J. L. (1977) The properties of a rat serum protein labelled by the injection of sodium selenite. Biochim. Biophys. Acta 500:61 70. HILL, K. E., YANG, J.-G. and BtSRK, R. F. (1988) Assessment of selenium intake: comparison of selenoprotein P concentration and glutathione peroxidase activity in rat plasma. F A S E B J. 2: A1088. HOEKSTRA,W. G. (1975) Biochemical function of selenium and its relation to vitamin E. Fed. Proc. 34: 2083-2089. MCCAV, P. B., GIBSON,D. D., FONG,K.-L. and HORNBROOK,K. R. (1976) Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochim. Biophys. Acta 431: 459-468. MERCURIO,S. D. and COMBS,G. F. (1986) Selenium-dependent glutathione peroxidase inhibitors increase toxicity of pro-oxidant compounds. J. Nutr. 116: 1726-1734. MOTCHNIK,P. A., GOMEZ,B. and TAPPEL,A. L. (1987) Rat plasma selenoprotein characterization and membrane receptor binding properties. Fed. Proc. 46: 907A. MOTSENBOCKER,M. A. and TAPPEL,A. L. (1982) A selenocysteine-containing selenium-transport protein in rat plasma. Biochim. Biophys. Acta 719: 147-153. OsmNo, N., JAMIESON,D. and CHANCE, B. (1975) The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver. Biochem. J. 146: 53~5. ROTRt;CK, J. T., POPE, A. L., GANTHER,H. E., SWANSON,A. B., HA~MAN, D. G. and HOEKSTRA,W. G. (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179: 585-590. SCHWARZ, K. and FOLTZ, C. M. (1957) Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: 3292-3293. SEVAN1AN,A., MUAKKASSAH-KELLV,S. F. and MONTESTRt;QUE,S. (1983) The influence of phospholipase A 2 and glutathione peroxidase on the elimination of membrane lipid peroxides. Arch. Biochem. Biophys. 223: 441-452. URSINt, F., MA1ORINO,M., VALENTE,M., FERRh L. and GREGOUN,C. (1982) Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochim. Biophys. Acta 710: 197-211. YANG, J.-G., MORRISON-PLUMrCmR,J. and Bt;RK, R. F. (1987) Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies. J. Biol. Chem. 262: 13372-13375. YANG, J.-G., HILt, K. E. and BURK, R. F. (1989) Dietary selenium intake controls rat plasma selenoprotein P concentration. J. Nutr. 119: 1010-1012.