- Email: [email protected]
Metal-Independent Putative Superoxide Dismutase Mimics in Chemistry, Biology, and Medicine
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO.
34, 141–144 (1996)
0055
Metal-Independent Putative Superoxide Dismutase Mimics in Chemistry, Biology, and Medicine NIHAL AHMAD, MINAKSHI MISRA, M. M. HUSAIN,1
AND
R. C. SRIVASTAVA2
Industrial Toxicology Research Centre, M. G. Marg, Lucknow 226001, India Received June 7, 1995 0
The excessive generation of superoxide radicals O2r with inadequate available defence provided by the enzyme superoxide dismutase (SOD) may result in the development and exacerbation of many of mankind’s common illnesses. The native SOD proves too problematic to be used for the prevention and cure of such diseases. A number of metal-independent synthetic SOD mimics, based on organic nitroxides, have been tried as therapeutic interventions. Among the widely studied mimics, 2-ethyl-2,5,5-trimethyl-3-oxazolidinanyl, 2,2,6,6-tetramethyl-1-piperidimyloxy, nitrosoureas, and triazene derivatives have indicated promising results with possible future applications in chemistry, biology, and medicine. q 1996 Academic Press, Inc.
INTRODUCTION
The list of reactive oxygen species (ROS) mediating pathological conditions continues to grow at such a rapid pace that even the superoxide dismutation appears to be a slower process. To date, no less than 50 pathological conditions are known to be mediated by free radical superoxide anion 0 (Or2 ) (Cross, 1987). The antioxidant enzyme superoxide dismutase (SOD) is a part of nature’s answer to this problem. SOD dismutes the superoxide radical by converting it into hydrogen peroxide and oxygen (McCord et al., 1969). The enzyme has received much attention and its application as a therapeutic intervention in various disease conditions has been attempted with limited success (Halliwell, 1984; Clark et al., 1985; Petkau, 1986; Flohe, 1988; Oda et al., 1989; Gerdes, 1985; Stern, 1982; Greenwald, 1990). There are several problems with the use of the enzyme SOD as a therapeutic agent. Because it has a very high molecular weight (30000–80000), it is prevented from entering into cells, thus providing only extracellular protection. In addition, the native enzyme is antigenic and too expensive to be used. Consequently, there has been considerable effort by a number of researchers to develop 1 To whom correspondence should be addressed at Industrial Toxicology Research Centre, Post Box No. 80, M. G. Marg, Lucknow 226001, India. 2 Present address: Human Nutrition Research Centre, USDA, Grand Forks, ND 58202-9034.
a stable, nontoxic, low-molecular-weight mimic to substitute for SOD that is inexpensive, permeable through cell membrane, and nonimmunogenic in nature. So far, a number of attempts have been made in this direction that have resulted in extensive studies on metal complexes. The complexes of copper, as potential SOD mimics, have been widely investigated (Kensler et al., 1988; Huber et al., 1987; Goldstein et al., 1990; Willingham and Sorenson, 1988, Dollwet et al., 1987; Yaping et al., 1992; Kimura et al., 1983; Mengchang et al., 1990; Sorenson, 1984; Jouini, 1986). This is probably because (i) it has long been known that in acidic medium Cu(II) behaves as an excellent catalyst for dismutation of 0 Or2 and (ii) the first SOD that was isolated contained copper (McCord, 1969). However, most of the Cu(II) complexes studied did not retain integrity and activity in the presence of the proteins in blood plasma or within cells. A number of complexes based on Mn and Fe have also been investigated with only limited success (Nagano et al., 1989; Darr et al., 1987; Itami et al., 1993; Weiss et al., 1993; Baudry et al., 1993). These metal complexes may prove to be effective in vitro but their application in vivo is questionable because the metal complexes easily dissociate, can catalyze many other harmful redox processes within the cell, exhibit high affinity toward proteins and amino acids, and may lose their activity upon binding to cellular components. Thus, some concrete and successful efforts have been made to develop metal-independent SOD mimics with proven applicability and therapeutic usefulness in preventing and/or curing the various 0 Or2 -mediated pathological conditions. 2-Ethyl-2,5,5-trimethyl-3-oxazolidinanyl (OXANO), 2,2,6,6Tetramethyl-1-piperidinyloxy (TEMPO), AND THEIR DERIVATIVES AS SOD MIMICS
Structure and Mechanism One particular compound, 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) was found to be oxidized 0 readily at pH 7.8 by O2r to 2-ethyl-2,5,5-trimethyl-3-oxazolidinanyl radical (OXANO) with a rate constant of 6.7 1 103 M01 sec01 and was stable. This redox system was employed 0 to detect O2r (Rosen et al., 1977) (Fig. 1).
141
AID
EES 1419
/
a105$$$121
07-18-96 02:34:22
0147-6513/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
eesa
AP: EES
142
AHMAD ET AL.
FIG. 1. OXANO/OXANOH redox system.
This reaction proved to be the basis for the use of OXANO as the first metal-independent superoxide dismutase mimic (Samuni et al., 1988). Samuni et al. (1988) established that superoxide can both reduce OXANO and oxidize OXANOH. They determined the rate constants for the reaction using two superoxide-generating systems: (i) xanthine–xanthine oxidase system and (ii) ionizing radiation. It was found that OXANOH oxidation as well as OXANO reduction were pH dependent, inhibitable by SOD, and resulted in a steadystate distribution of OXANO and OXANOH independent of their initial concentrations. Thus, the OXANO/OXANOH couple was found to exhibit a metal-independent SOD-like activity. OXANO is soluble in both polar and nonpolar solvents, relatively stable within cells, nontoxic in millimolar 0 range, might substitute for SOD in protecting against O2r inside as well as outside the cell, and, with suitable structural modification, can be improved in efficacy/activity. These preliminary findings from Samuni et al, (1988) on the possible use of stable nitroxide radicals as SOD mimics opened an area of immense interest for chemists and biochemists. A study on synthesis and evaluation of SOD-mimicing ability of nitroxides soon followed in which a series of five-membered and six-membered ring nitroxides were 0 studied for their abilities to scavenge O2r (Mitchell et al., 1990). The five-membered ring nitroxides were based on OXANO with structural modification (Fig. 2). The six-membered ring nitroxides that have extensively been studied are (i) 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and (ii) 4-hydroxy-2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPOL), which is a derivative of TEMPO (Fig. 3). Protective Role The utility of OXANO, TEMPO, and their derivatives in protecting mammalian cells against oxidative stress has been
FIG. 2. OXANO and its derivatives.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
FIG. 3. TEMPO and its derivative.
evaluated using two different means: (i) protection from hypoxanthine–xanthine oxidase-induced killing of Chinese hamster V79 cells, and (ii) protection from H2O2-induced killing of Chinese hamster V79 cells. The nitroxides were found to act against oxidative damage effectively to behave as efficient SOD mimics. These nitroxide SOD mimics were also found to oxidise DNA–Fe2/ rapidly,thus interrupting Fenton reaction to prevent the formation of •OH radicals and/or higher oxidation of metal ions. DNA–Fe2/ / RR*N• –O / H/ r DNA–Fe3/ / RR*NOH The role of ROS in bleomycin-induced gene mutation in Chinese hamster ovary (CHO) cells is now established. This mutagenesis in mammalian cells is considerably inhibited by 1-hr TEMPOL (1 mM) pretreatment giving further support in favor of its ability to function as a SOD mimic (An and Hsie, 1992). TEMPOL also provides complete protection to the CHO AS 52 cells from the cytotoxic and mutagenic effects of the hypoxanthine – xanthine oxidase system and hydrogen peroxide (DeGraff et al., 1992). TEMPOL provided protection to the cultured rabbit lens epithelial cells from hydrogen peroxide insult (Mitchell et al., 1991). It prevented the H2O2-induced inhibition of cell growth, blebbing of cell membrane, the decrease in NAD/, the activation of (poly)ADP-ribose polymerase (an enzyme involved in DNA repair), and limited the induction of single strand breaks in DNA normally brought about by H2O2 . TEMPOL, however, did not prevent the H2O2-induced decrease in reduced glutathione, lactate production, the activity of glyceraldehyde 3-phosphate dehydrogenase, and the increase in oxidized glutathione and hexose monophosphate shunt activity. A study to delineate the mechanism of action for these SOD mimics revealed that the inhibition of oxidative damage by metal-free as well as metal-based SOD mimics is attributed to either SOD mimic reacting with reduced transition metal to block the Fenton’s reaction and/or intercepting and detoxifying intracellular organic free radicals. TEMPOL also protected the mammalian cells against oxygen-dependent radiation-induced damage (Mitchell et al., 1991). The pretreatment of Chinese hamster cells under aerobic conditions with 5–100 mM TEMPOL resulted in appreciable protection against X rays. TEMPOL treatment under
eesa
AP: EES
143
METAL-INDEPENDENT SOD MIMICS
It has also been evaluated that these compounds have high antitumor activity and low toxicity and the beneficial effects are attributed to the antioxidant effect of the incorporated nitroxide, derived efficiently from the redox cycling reaction. CONCLUSION
It can be concluded that the metal-independent SOD mimics that protect against a number of oxidative insults are well tolerated by mammalian cells and may be used (i) as basic 0 biological probes in studying the effect of O2r within the cells, and (ii) to delineate the intracellular mechanism of the processes mediating the damages through oxygen metabolites. These findings may have a wide range of applications in chemistry, biology, and medicine. ACKNOWLEDGMENT The authors are thankful to Dr. R. C. Srimal, Director, for encouragement during this work.
REFERENCES FIG. 4. Nitrosoureas and triazenes.
hypoxic conditions, however, did not provide any radioprotection and the exact mechanism(s) for these events is yet to be elucidated. TEMPOL was found to protect against tumor necrosis factor (TNF) cytoxicity. The WEHI or L929 cells on incubating with TNF and TEMPOL under different conditions revealed that this stable nitroxide significantly increases the survival of the cells (Pogrebniak et al., 1991). This finding may prove to be useful in conditions associated with free radical lymphokine interactions such as ischemia reperfusion, oxygen toxicity, and sepsis. A couple of studies have found the ability of TEMPOL as an efficient SOD mimic to protect against bleomycin mutagenesis, possibly mediated 0 through the free radical O2r , in mammalian cells (An and Hsie, 1993, 1994). NITROSOUREAS AND TRIAZENE DERIVATIVES AS SOD MIMICS
The nitrosoureas and triazenes are proven active alkylating chemotherapeutic agents used for the treatment of a number of clinical neoplasm (Carter et al., 1972; Shealy, 1970). Very recently, the superoxide scavenging activities of some novel spin-labeled nitrosourea and triazene derivatives have been investigated that were found to possess excellent superoxide scavenging activities (Gadzheva et al., 1994). The various compounds that have been investigated are provided in Fig. 4.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
An, J., and Hsie, A. W. (1992). Effects of an inhibitor and a mimic of superoxide dismutase on bleomyein mutagenesis in chinese hamster ovary cells. Mutat. Res. 270, 167–175. An, J., and Hsie, A. W. (1993). Polymerase chain reaction based deletion screening of bleomycin induced 6-thio-guanine-resistant mutants in chinese hamster ovary cells: The effects of an inhibitor and a mimic of superoxide dismutase. Mutat. Res. 289, 215–222. An, J., and Hsie, A. W., (1994). Polymerase chain reaction directed DNA sequencing of bleomycin induced nondeletion type, 6-thioguanine resistant mutants in chinese hamster ovary cell derivative AS 52: effects of an inhibitor and a mimic of superoxide dismutase. Environ. Mol. Mutagen. 23, 101–109. Baudry, M., Etienne, S., Bruce, A., Palucki, M., Jacobsen, E., and Malfroy, B. (1993). Salen-manganese complexes are superoxide dismutase mimics. Biochem. Biophys. Res. Commun. 192, 964–968. Carter, S. K., Schabel, F. M., Broder, L. E., and Johnston, J. P., (1972). 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and other nitrososureas in cancer treatment. Adv. Cancer Res. 16, 273–332. Clark, I., Crowden, W., and Hunt, N. (1985). Free radical induced pathology. Med. Res. Rev. 5, 297–332. Cross, C. (1987). Oxygen radicals and human disease. Ann. Intern. Med. 107, 526–545. Darr, D., Zarilla, K. A., and Fridovich, I. (1987). A mimic of superoxide dismutase activity based upon desferrioxamine-B and manganese (IV). Arch. Biochem. Biophys. 258, 351–355. DeGraff, W. G., Krishna, C. M., Russo, A., and Mitchell, J. B. (1992). Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens. Environ. Mol. Mutagen. 19, 21–26. Dollwet, H. H. A., McNicholas, J. B., Pezesk, A., and Sorenson, J. R. J. (1987). Superoxide dimutase mimetic activity of antiepileptic drug copper complexes. Trace Elem. Med. 4, 13–20. Flohe, L. (1988). Superoxide dismutase for therapeutic use: Clinical experience, dead ends, and hope. Mol. Cell. Biochem. 84, 123–131. Gadzheva, V., Ichimori, K., Nakazawa, H., and Raikov, Z. (1994). Superox-
eesa
AP: EES
144
AHMAD ET AL.
ide scavenging activity of spin-labeled nitrosourea and triazene derivatives. Free Rad. Res. 21, 177–186. Gerdes, J. S. (1985). Superoxide dismutase in prevention of bronchopulmonary dysplasia. J. Pediatr. 106, 1057–1061. Goldstein, S., Czapski, G., and Meyerstein, D. (1990). A mechanistic study of the copper (II)-peptide catalysed superoxide dismutation. A Pulse radiolysis study. J. Am. Chem. Soc. 112, 6489–6492. Greenwald, R. A. (1990). Superoxide dismutase and catalase as therapeutic agents for human diseases. Free Rad. Biol. Med. 8, 201–209. Halliwell, B. (1984). Oxygen radicals: A commensense look at their nature and medical importance. Med. Biol. 62, 71–77. Huber, K. R., Sridhar, R., Griffith, E. H., Amma, E. L., and Roberts, J. (1987). Superoxide dismutase like activities of copper (II) complexes tested in serum. Biochim. Biophys. Acta 915, 267–276. Itami, C., Matsunaga, H., Sawada, T., Sakurai, H., and Kimura, Y. (1993). Superoxide dismutase mimetic activities of metal complexes of l-2(2pyridyl)-1-pyrroline-5-carboxylic acid (pyrimine). Biochem. Biophys. Res. Commun. 197, 536–541. Jouini, M., Laplaye, G., Hult, J., Julien, R., and Ferradini, C. (1986). Catalytic activity of copper (II) oxidized glutathione complex on aqueous superoxide ion dismutation. J. Inorg. Biochem. 26, 269–280. Kensler, T. W., Bush, D. M., and Kozumbo, W. J. (1988). Inhibition of tumor promotion by a biomimetic superoxide dismutase. Science 221, 75–77. Kimura, E., Yatsunami, A., Watanabe, A., Machida, P., Koike, T., Fujioke, H., Kuramoto, Y., Sumomogi, M., Kunimitsu, K., and Yamashita, A. (1983). Further studies on superoxide dismutase activities macrocyclic polyamine complexes of copper (II). Biochim. Biophys. Acta 745, 37– 41. McCord, J. M., and Fridovich, I. (1969). Superoxide dismutase, an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 22, 6049– 6055. Mengchang, S., Qinhui, L., Shourong, Z., Qingyun, T., and Anbang, D. (1990). A Kinetic study on dismutation of superoxide ion by macrocyclic dioxotetramine copper (II) complex by pulse radiolysis. Chem. J. Chinese Univ. 11, 1322–1326. Mitchell, J. B., Degraff, W. B., Kaufman, D., Krishna, C. M., Samuni, A., Finkelstein, F., Ahn, M. S., Hahn, S. M., Gamson, J., and Russo, A.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
(1991). Inhibition of oxygen dependent radiation induced damage by the nitroxide superoxide dismutase mimic. Tempol. Arch. Biochem. Biophys. 289, 62–70. Mitchell, J. B., Samuni, A., Krishna, C. M., Degraff, W. G., Ahn, M. S., Samuni, U., and Russo, A. (1990). Biologically active metal independent superoxide dismutale mimics. Biochemistry 29, 2802–2807. Nagano, T., Hirano, T., and Hirobe, M. (1989). Superoxide dismutase mimics based on iron in vivo. J. Biol. Chem. 264, 9243–9249. Oda, T., Akaike, T., Hamamoto, T., Suzuki, F., Hirano, T., and Maeda, H. (1989). Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer conjugated SOD. Science 244, 974–976. Petkau, A. (1986). Scientific basis for the clinical use of superoxide dismutase. Cancer Treat. Rev. 13, 17–44. Pogrebniak, H., Mathews, W., Mitchell, J., Russo, A., Samuni, A., and Pass, H., (1991). Spin trap protection from tumor necrosis factor cytotoxicity. J. Surg. Res. 50, 469–474. Rosen, G. M., Rauckman, E. J., and Hanck, K. W. (1977). Selective bioreduction of nitroxides by rat liver microsomes. Toxicol. Lett. 1, 71–74. Samuni, A., Krishna, C. M., Riesz, P., Finkelstein, E., and Russo, A. (1988). A novel metal-free low molecular weight superoxide dismutase mimic. J. Biol. Chem. 263, 17921–17924. Shealy, Y. F. (1970). Synthesis and biological activity of 5-aminoimidazoles and 5-triazenoimidazoles. J. Pharm. Sci. 59, 1533–1558. Sorenson, J. R. J. (1984). Bis(3,5-diisopropylsalicylato) copper (II), a potent radioprotectant with superoxide dismutase mimetic activity. J. Med. Chem. 27, 1747–1749. Stern, L. Z., Ringel, S. P., Ziter, F. A., Ionasescu, V., Pellegrino, R. J., and Snyder, R. D. (1982). Drug trial of Superoxide dismutase in Duchenne’s muscular dystrophy. Arch. Neurol. 39, 342–346. Weiss, R. H., Flickinger, A. J., Rivers, W. J., Hardy, M. M., Aston, K. W., Ryan, U. S., and Riley, D. P. (1993). Evaluation of activity of Putative superoxide dismutase mimics. J. Biol. Chem. 268, 23049–23054. Willingham, W. M., and Sorenson, J. R. J. (1988). Copper (II) ethylenediaminetetracetate does disproportionate superoxide. Biochem. Biophys. Res. Commun. 150, 252–258. Yaping, T., Yunzhong, F., Qinhui, L., Mengchang, S., Qin, L., and Wenmei, S. (1992). The inhibitory effects of 21 mimics of superoxide dismutase on luminol mediated chemiluminescence emitted from PMA-stimulated polymorphonuclear leukocyte. Free Rad. Biol. Med. 13, 533–541.
eesa
AP: EES
34, 141–144 (1996)
0055
Metal-Independent Putative Superoxide Dismutase Mimics in Chemistry, Biology, and Medicine NIHAL AHMAD, MINAKSHI MISRA, M. M. HUSAIN,1
AND
R. C. SRIVASTAVA2
Industrial Toxicology Research Centre, M. G. Marg, Lucknow 226001, India Received June 7, 1995 0
The excessive generation of superoxide radicals O2r with inadequate available defence provided by the enzyme superoxide dismutase (SOD) may result in the development and exacerbation of many of mankind’s common illnesses. The native SOD proves too problematic to be used for the prevention and cure of such diseases. A number of metal-independent synthetic SOD mimics, based on organic nitroxides, have been tried as therapeutic interventions. Among the widely studied mimics, 2-ethyl-2,5,5-trimethyl-3-oxazolidinanyl, 2,2,6,6-tetramethyl-1-piperidimyloxy, nitrosoureas, and triazene derivatives have indicated promising results with possible future applications in chemistry, biology, and medicine. q 1996 Academic Press, Inc.
INTRODUCTION
The list of reactive oxygen species (ROS) mediating pathological conditions continues to grow at such a rapid pace that even the superoxide dismutation appears to be a slower process. To date, no less than 50 pathological conditions are known to be mediated by free radical superoxide anion 0 (Or2 ) (Cross, 1987). The antioxidant enzyme superoxide dismutase (SOD) is a part of nature’s answer to this problem. SOD dismutes the superoxide radical by converting it into hydrogen peroxide and oxygen (McCord et al., 1969). The enzyme has received much attention and its application as a therapeutic intervention in various disease conditions has been attempted with limited success (Halliwell, 1984; Clark et al., 1985; Petkau, 1986; Flohe, 1988; Oda et al., 1989; Gerdes, 1985; Stern, 1982; Greenwald, 1990). There are several problems with the use of the enzyme SOD as a therapeutic agent. Because it has a very high molecular weight (30000–80000), it is prevented from entering into cells, thus providing only extracellular protection. In addition, the native enzyme is antigenic and too expensive to be used. Consequently, there has been considerable effort by a number of researchers to develop 1 To whom correspondence should be addressed at Industrial Toxicology Research Centre, Post Box No. 80, M. G. Marg, Lucknow 226001, India. 2 Present address: Human Nutrition Research Centre, USDA, Grand Forks, ND 58202-9034.
a stable, nontoxic, low-molecular-weight mimic to substitute for SOD that is inexpensive, permeable through cell membrane, and nonimmunogenic in nature. So far, a number of attempts have been made in this direction that have resulted in extensive studies on metal complexes. The complexes of copper, as potential SOD mimics, have been widely investigated (Kensler et al., 1988; Huber et al., 1987; Goldstein et al., 1990; Willingham and Sorenson, 1988, Dollwet et al., 1987; Yaping et al., 1992; Kimura et al., 1983; Mengchang et al., 1990; Sorenson, 1984; Jouini, 1986). This is probably because (i) it has long been known that in acidic medium Cu(II) behaves as an excellent catalyst for dismutation of 0 Or2 and (ii) the first SOD that was isolated contained copper (McCord, 1969). However, most of the Cu(II) complexes studied did not retain integrity and activity in the presence of the proteins in blood plasma or within cells. A number of complexes based on Mn and Fe have also been investigated with only limited success (Nagano et al., 1989; Darr et al., 1987; Itami et al., 1993; Weiss et al., 1993; Baudry et al., 1993). These metal complexes may prove to be effective in vitro but their application in vivo is questionable because the metal complexes easily dissociate, can catalyze many other harmful redox processes within the cell, exhibit high affinity toward proteins and amino acids, and may lose their activity upon binding to cellular components. Thus, some concrete and successful efforts have been made to develop metal-independent SOD mimics with proven applicability and therapeutic usefulness in preventing and/or curing the various 0 Or2 -mediated pathological conditions. 2-Ethyl-2,5,5-trimethyl-3-oxazolidinanyl (OXANO), 2,2,6,6Tetramethyl-1-piperidinyloxy (TEMPO), AND THEIR DERIVATIVES AS SOD MIMICS
Structure and Mechanism One particular compound, 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) was found to be oxidized 0 readily at pH 7.8 by O2r to 2-ethyl-2,5,5-trimethyl-3-oxazolidinanyl radical (OXANO) with a rate constant of 6.7 1 103 M01 sec01 and was stable. This redox system was employed 0 to detect O2r (Rosen et al., 1977) (Fig. 1).
141
AID
EES 1419
/
a105$$$121
07-18-96 02:34:22
0147-6513/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
eesa
AP: EES
142
AHMAD ET AL.
FIG. 1. OXANO/OXANOH redox system.
This reaction proved to be the basis for the use of OXANO as the first metal-independent superoxide dismutase mimic (Samuni et al., 1988). Samuni et al. (1988) established that superoxide can both reduce OXANO and oxidize OXANOH. They determined the rate constants for the reaction using two superoxide-generating systems: (i) xanthine–xanthine oxidase system and (ii) ionizing radiation. It was found that OXANOH oxidation as well as OXANO reduction were pH dependent, inhibitable by SOD, and resulted in a steadystate distribution of OXANO and OXANOH independent of their initial concentrations. Thus, the OXANO/OXANOH couple was found to exhibit a metal-independent SOD-like activity. OXANO is soluble in both polar and nonpolar solvents, relatively stable within cells, nontoxic in millimolar 0 range, might substitute for SOD in protecting against O2r inside as well as outside the cell, and, with suitable structural modification, can be improved in efficacy/activity. These preliminary findings from Samuni et al, (1988) on the possible use of stable nitroxide radicals as SOD mimics opened an area of immense interest for chemists and biochemists. A study on synthesis and evaluation of SOD-mimicing ability of nitroxides soon followed in which a series of five-membered and six-membered ring nitroxides were 0 studied for their abilities to scavenge O2r (Mitchell et al., 1990). The five-membered ring nitroxides were based on OXANO with structural modification (Fig. 2). The six-membered ring nitroxides that have extensively been studied are (i) 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and (ii) 4-hydroxy-2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPOL), which is a derivative of TEMPO (Fig. 3). Protective Role The utility of OXANO, TEMPO, and their derivatives in protecting mammalian cells against oxidative stress has been
FIG. 2. OXANO and its derivatives.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
FIG. 3. TEMPO and its derivative.
evaluated using two different means: (i) protection from hypoxanthine–xanthine oxidase-induced killing of Chinese hamster V79 cells, and (ii) protection from H2O2-induced killing of Chinese hamster V79 cells. The nitroxides were found to act against oxidative damage effectively to behave as efficient SOD mimics. These nitroxide SOD mimics were also found to oxidise DNA–Fe2/ rapidly,thus interrupting Fenton reaction to prevent the formation of •OH radicals and/or higher oxidation of metal ions. DNA–Fe2/ / RR*N• –O / H/ r DNA–Fe3/ / RR*NOH The role of ROS in bleomycin-induced gene mutation in Chinese hamster ovary (CHO) cells is now established. This mutagenesis in mammalian cells is considerably inhibited by 1-hr TEMPOL (1 mM) pretreatment giving further support in favor of its ability to function as a SOD mimic (An and Hsie, 1992). TEMPOL also provides complete protection to the CHO AS 52 cells from the cytotoxic and mutagenic effects of the hypoxanthine – xanthine oxidase system and hydrogen peroxide (DeGraff et al., 1992). TEMPOL provided protection to the cultured rabbit lens epithelial cells from hydrogen peroxide insult (Mitchell et al., 1991). It prevented the H2O2-induced inhibition of cell growth, blebbing of cell membrane, the decrease in NAD/, the activation of (poly)ADP-ribose polymerase (an enzyme involved in DNA repair), and limited the induction of single strand breaks in DNA normally brought about by H2O2 . TEMPOL, however, did not prevent the H2O2-induced decrease in reduced glutathione, lactate production, the activity of glyceraldehyde 3-phosphate dehydrogenase, and the increase in oxidized glutathione and hexose monophosphate shunt activity. A study to delineate the mechanism of action for these SOD mimics revealed that the inhibition of oxidative damage by metal-free as well as metal-based SOD mimics is attributed to either SOD mimic reacting with reduced transition metal to block the Fenton’s reaction and/or intercepting and detoxifying intracellular organic free radicals. TEMPOL also protected the mammalian cells against oxygen-dependent radiation-induced damage (Mitchell et al., 1991). The pretreatment of Chinese hamster cells under aerobic conditions with 5–100 mM TEMPOL resulted in appreciable protection against X rays. TEMPOL treatment under
eesa
AP: EES
143
METAL-INDEPENDENT SOD MIMICS
It has also been evaluated that these compounds have high antitumor activity and low toxicity and the beneficial effects are attributed to the antioxidant effect of the incorporated nitroxide, derived efficiently from the redox cycling reaction. CONCLUSION
It can be concluded that the metal-independent SOD mimics that protect against a number of oxidative insults are well tolerated by mammalian cells and may be used (i) as basic 0 biological probes in studying the effect of O2r within the cells, and (ii) to delineate the intracellular mechanism of the processes mediating the damages through oxygen metabolites. These findings may have a wide range of applications in chemistry, biology, and medicine. ACKNOWLEDGMENT The authors are thankful to Dr. R. C. Srimal, Director, for encouragement during this work.
REFERENCES FIG. 4. Nitrosoureas and triazenes.
hypoxic conditions, however, did not provide any radioprotection and the exact mechanism(s) for these events is yet to be elucidated. TEMPOL was found to protect against tumor necrosis factor (TNF) cytoxicity. The WEHI or L929 cells on incubating with TNF and TEMPOL under different conditions revealed that this stable nitroxide significantly increases the survival of the cells (Pogrebniak et al., 1991). This finding may prove to be useful in conditions associated with free radical lymphokine interactions such as ischemia reperfusion, oxygen toxicity, and sepsis. A couple of studies have found the ability of TEMPOL as an efficient SOD mimic to protect against bleomycin mutagenesis, possibly mediated 0 through the free radical O2r , in mammalian cells (An and Hsie, 1993, 1994). NITROSOUREAS AND TRIAZENE DERIVATIVES AS SOD MIMICS
The nitrosoureas and triazenes are proven active alkylating chemotherapeutic agents used for the treatment of a number of clinical neoplasm (Carter et al., 1972; Shealy, 1970). Very recently, the superoxide scavenging activities of some novel spin-labeled nitrosourea and triazene derivatives have been investigated that were found to possess excellent superoxide scavenging activities (Gadzheva et al., 1994). The various compounds that have been investigated are provided in Fig. 4.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
An, J., and Hsie, A. W. (1992). Effects of an inhibitor and a mimic of superoxide dismutase on bleomyein mutagenesis in chinese hamster ovary cells. Mutat. Res. 270, 167–175. An, J., and Hsie, A. W. (1993). Polymerase chain reaction based deletion screening of bleomycin induced 6-thio-guanine-resistant mutants in chinese hamster ovary cells: The effects of an inhibitor and a mimic of superoxide dismutase. Mutat. Res. 289, 215–222. An, J., and Hsie, A. W., (1994). Polymerase chain reaction directed DNA sequencing of bleomycin induced nondeletion type, 6-thioguanine resistant mutants in chinese hamster ovary cell derivative AS 52: effects of an inhibitor and a mimic of superoxide dismutase. Environ. Mol. Mutagen. 23, 101–109. Baudry, M., Etienne, S., Bruce, A., Palucki, M., Jacobsen, E., and Malfroy, B. (1993). Salen-manganese complexes are superoxide dismutase mimics. Biochem. Biophys. Res. Commun. 192, 964–968. Carter, S. K., Schabel, F. M., Broder, L. E., and Johnston, J. P., (1972). 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and other nitrososureas in cancer treatment. Adv. Cancer Res. 16, 273–332. Clark, I., Crowden, W., and Hunt, N. (1985). Free radical induced pathology. Med. Res. Rev. 5, 297–332. Cross, C. (1987). Oxygen radicals and human disease. Ann. Intern. Med. 107, 526–545. Darr, D., Zarilla, K. A., and Fridovich, I. (1987). A mimic of superoxide dismutase activity based upon desferrioxamine-B and manganese (IV). Arch. Biochem. Biophys. 258, 351–355. DeGraff, W. G., Krishna, C. M., Russo, A., and Mitchell, J. B. (1992). Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens. Environ. Mol. Mutagen. 19, 21–26. Dollwet, H. H. A., McNicholas, J. B., Pezesk, A., and Sorenson, J. R. J. (1987). Superoxide dimutase mimetic activity of antiepileptic drug copper complexes. Trace Elem. Med. 4, 13–20. Flohe, L. (1988). Superoxide dismutase for therapeutic use: Clinical experience, dead ends, and hope. Mol. Cell. Biochem. 84, 123–131. Gadzheva, V., Ichimori, K., Nakazawa, H., and Raikov, Z. (1994). Superox-
eesa
AP: EES
144
AHMAD ET AL.
ide scavenging activity of spin-labeled nitrosourea and triazene derivatives. Free Rad. Res. 21, 177–186. Gerdes, J. S. (1985). Superoxide dismutase in prevention of bronchopulmonary dysplasia. J. Pediatr. 106, 1057–1061. Goldstein, S., Czapski, G., and Meyerstein, D. (1990). A mechanistic study of the copper (II)-peptide catalysed superoxide dismutation. A Pulse radiolysis study. J. Am. Chem. Soc. 112, 6489–6492. Greenwald, R. A. (1990). Superoxide dismutase and catalase as therapeutic agents for human diseases. Free Rad. Biol. Med. 8, 201–209. Halliwell, B. (1984). Oxygen radicals: A commensense look at their nature and medical importance. Med. Biol. 62, 71–77. Huber, K. R., Sridhar, R., Griffith, E. H., Amma, E. L., and Roberts, J. (1987). Superoxide dismutase like activities of copper (II) complexes tested in serum. Biochim. Biophys. Acta 915, 267–276. Itami, C., Matsunaga, H., Sawada, T., Sakurai, H., and Kimura, Y. (1993). Superoxide dismutase mimetic activities of metal complexes of l-2(2pyridyl)-1-pyrroline-5-carboxylic acid (pyrimine). Biochem. Biophys. Res. Commun. 197, 536–541. Jouini, M., Laplaye, G., Hult, J., Julien, R., and Ferradini, C. (1986). Catalytic activity of copper (II) oxidized glutathione complex on aqueous superoxide ion dismutation. J. Inorg. Biochem. 26, 269–280. Kensler, T. W., Bush, D. M., and Kozumbo, W. J. (1988). Inhibition of tumor promotion by a biomimetic superoxide dismutase. Science 221, 75–77. Kimura, E., Yatsunami, A., Watanabe, A., Machida, P., Koike, T., Fujioke, H., Kuramoto, Y., Sumomogi, M., Kunimitsu, K., and Yamashita, A. (1983). Further studies on superoxide dismutase activities macrocyclic polyamine complexes of copper (II). Biochim. Biophys. Acta 745, 37– 41. McCord, J. M., and Fridovich, I. (1969). Superoxide dismutase, an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 22, 6049– 6055. Mengchang, S., Qinhui, L., Shourong, Z., Qingyun, T., and Anbang, D. (1990). A Kinetic study on dismutation of superoxide ion by macrocyclic dioxotetramine copper (II) complex by pulse radiolysis. Chem. J. Chinese Univ. 11, 1322–1326. Mitchell, J. B., Degraff, W. B., Kaufman, D., Krishna, C. M., Samuni, A., Finkelstein, F., Ahn, M. S., Hahn, S. M., Gamson, J., and Russo, A.
AID
EES 1419
/
a105$$$122
07-18-96 02:34:22
(1991). Inhibition of oxygen dependent radiation induced damage by the nitroxide superoxide dismutase mimic. Tempol. Arch. Biochem. Biophys. 289, 62–70. Mitchell, J. B., Samuni, A., Krishna, C. M., Degraff, W. G., Ahn, M. S., Samuni, U., and Russo, A. (1990). Biologically active metal independent superoxide dismutale mimics. Biochemistry 29, 2802–2807. Nagano, T., Hirano, T., and Hirobe, M. (1989). Superoxide dismutase mimics based on iron in vivo. J. Biol. Chem. 264, 9243–9249. Oda, T., Akaike, T., Hamamoto, T., Suzuki, F., Hirano, T., and Maeda, H. (1989). Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer conjugated SOD. Science 244, 974–976. Petkau, A. (1986). Scientific basis for the clinical use of superoxide dismutase. Cancer Treat. Rev. 13, 17–44. Pogrebniak, H., Mathews, W., Mitchell, J., Russo, A., Samuni, A., and Pass, H., (1991). Spin trap protection from tumor necrosis factor cytotoxicity. J. Surg. Res. 50, 469–474. Rosen, G. M., Rauckman, E. J., and Hanck, K. W. (1977). Selective bioreduction of nitroxides by rat liver microsomes. Toxicol. Lett. 1, 71–74. Samuni, A., Krishna, C. M., Riesz, P., Finkelstein, E., and Russo, A. (1988). A novel metal-free low molecular weight superoxide dismutase mimic. J. Biol. Chem. 263, 17921–17924. Shealy, Y. F. (1970). Synthesis and biological activity of 5-aminoimidazoles and 5-triazenoimidazoles. J. Pharm. Sci. 59, 1533–1558. Sorenson, J. R. J. (1984). Bis(3,5-diisopropylsalicylato) copper (II), a potent radioprotectant with superoxide dismutase mimetic activity. J. Med. Chem. 27, 1747–1749. Stern, L. Z., Ringel, S. P., Ziter, F. A., Ionasescu, V., Pellegrino, R. J., and Snyder, R. D. (1982). Drug trial of Superoxide dismutase in Duchenne’s muscular dystrophy. Arch. Neurol. 39, 342–346. Weiss, R. H., Flickinger, A. J., Rivers, W. J., Hardy, M. M., Aston, K. W., Ryan, U. S., and Riley, D. P. (1993). Evaluation of activity of Putative superoxide dismutase mimics. J. Biol. Chem. 268, 23049–23054. Willingham, W. M., and Sorenson, J. R. J. (1988). Copper (II) ethylenediaminetetracetate does disproportionate superoxide. Biochem. Biophys. Res. Commun. 150, 252–258. Yaping, T., Yunzhong, F., Qinhui, L., Mengchang, S., Qin, L., and Wenmei, S. (1992). The inhibitory effects of 21 mimics of superoxide dismutase on luminol mediated chemiluminescence emitted from PMA-stimulated polymorphonuclear leukocyte. Free Rad. Biol. Med. 13, 533–541.
eesa
AP: EES