Apoptosis and Free Radicals

BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.

59, 93–97 (1996)

0072

MINIREVIEW Apoptosis and Free Radicals IRINA STOIAN,* ANDRA OROS,†

AND

ELENA MOLDOVEANU†

*Department of Biochemistry, UMF, Bucharest, Romania; and †Victor Babes Institute, Bucharest, 76201 Romania Received May 31, 1996

in cellular macromolecules: lipid peroxidation, DNA damage, and enzyme inactivation. The latter occurs because residues crucial for protein functions, such as methionine, cysteine, histidine, and tryptophan, are the main targets for ROS. NO induces inhibition of mitochondrial respiration and DNA synthesis by binding to iron and inhibiting several iron-containing enzymes (1). The relationship between free radicals and apoptosis is suggested by many experimental findings. Apoptosis can be induced by free radicals generated outside or inside the cells: H2O2 and menadione can induce apoptosis in a murine cell line (2); Snitrosocysteine (generating NO) and 3-morpholinosydonimine (generating NO and O20) cause apoptosis in cortical cell culture (3); and monocyte-derived H2O2 induces apoptosis in NK cells (4). Enzymes usually activated by reactive oxygen species and free radical scavengers can suppress apoptosis induced by factors that are not in themselves oxidants: apoptosis induced in ovarian follicles deprived of follicle-stimulating hormone (FSH) can be inhibited by superoxide dismutase (SOD), ascorbic acid, and N-acetyl-L-cysteine (NAC) (5); nitrone spin traps and a nitroxide antioxidant inhibit apoptosis induced by glucocorticoids in thymocytes (6); and overexpression of glutathione peroxidase can protect interleukin-3-dependent murine cells against apoptosis induced by IL-3 deprivation (2). The Bcl-2 gene (homologous to ced 9 in nematodes) encodes a protein that can suppress cell death and block effects of oxygen free radicals, acting as an antioxidant or free radical scavenger (2), providing additional evidence linking free radicals and apoptosis. Because reactive oxygen species are involved in

Free radicals that appear during physiological processes may lead to apoptosis in some pathological conditions when antioxidant capacity of the tissue is surpassed. Additionally, free radicals are involved in the control of apoptosis; antioxidant agents suppress apoptosis induced by a variety of stimuli. The possibility that apoptosis is regulated by modulation of the levels of free radicals is discussed. q 1996 Academic Press, Inc.

Apoptosis is a physiologic mode of cell death. Although the signals inducing this type of cell death in various cell types are very different, the characteristic morphological features are the same for cells undergoing apoptosis: nuclear condensation, membrane blebbing, and formation of apoptotic bodies. These common characteristics suggest a common biochemical mechanism regardless of the cell or signal type. However, the details of the biochemical steps underlying this mechanism are yet unknown. Recently, it has been suggested that apoptosis may be caused by the accumulation of free radicals. Free radicals appear during physiological processes, including oxidation–reduction reactions such as ubiquinone oxidation in mitochondria (O20); cytochrome P450 oxidation in microsomal membranes, related to the metabolism of foreign substances (O20 , singlet 1 O2); peroxysomal activity (H2O2); reaction of O20 with H2O2 in the catalytic presence of loosely bound iron (HO0, •HO); prostaglandin synthesis; respiratory burst of phagocytic cells such as neutrophils, macrophages, and monocytes; the impact of irradiation; xanthine oxidase activity; and arginine oxidation by a family of nitric oxide (NO) synthases. Reactive oxygen species (ROS) induce alterations 93

1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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various biologic processes, including activation of gene expression, regulation of proliferative events, and cellular response to cytokines, it was suggested that oxidative stress may be a mediator of apoptosis (7). This speculation assumes that oxygen free radicals not only induce expression of antioxidant defense or of repair proteins, but also exert a signaling role in normal cellular functions. METAL IONS AND THEIR IMPLICATIONS IN APOPTOSIS Many studies report that an increasing influx of calcium precedes apoptosis (8–10). In general, rising cytoplasmic free calcium levels are associated with the generation of reactive oxygen species. The permeability of the membrane to substances that do not normally cross it (Ca2/) may be caused by lipid peroxidation (11). Lipid peroxidation also was reported to increase after exposure of the cell to apoptotic stimulus (2). A recent study (12) reports the specific site of action of oxygen radicals on calmodulin to be one of the vicinal methionines near the C-terminus of calmodulin. This oxidation modifies the affinity of calmodulin for calcium and leads to the loss of its ability to activate plasma membrane Ca-ATPase, which is activated by calmodulin to maintain the low intracellular Ca2/ concentration. Under physiological conditions, oxidized methionine is reduced by methionine sulfoxide reductase cooperating with thioredoxin (see below). The disruption of intracellular Ca2/ homeostasis can cause activation of various Ca2/-dependent enzymes, including phospholipases, proteases, transglutaminases, NO synthetases, and endonucleases, which can lead to cell death. The activation of phospholipases by calcium and the accumulation of fatty acids together with the activation of lipid peroxidation induce changes in the lipid bilayers and play a role in the conversion of reversible tissue damage into irreversible lesions. Additionally, the activation of calcium-dependent nonlysosomal proteases seems to be somehow responsible for cell death and may play a role in bleb formation on the membrane surface (13). Calcium is not the sole cation regulatory signal for endonuclease activation; endonuclease activity is regulated by other cations, including Mg2/, Na/, and Zn2/. It has been presumed that the endonuclease contains a Zn2/ binding site and that Zn2/ inactivates the enzyme by competing with Ca2/ either competitively or allosterically (14). Zinc is generally

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reported to inhibit apoptosis by inhibiting specific endonucleases. It is also very possible that this inhibition may be the result of Zn2/ inhibition of lipid peroxidation (11). Reactions of copper ions with H2O2 seem to generate •OH (Fenton reaction). Apoptosis induced in thymocytes by dexamethasone and etoposide was inhibited by copper chelators, suggesting that a Fenton reaction is a possible stage in apoptosis. Iron can also undergo a Fenton reaction to generate •OH. In the same experiment, iron chelators had no effect (15). ROLES OF CELLULAR THIOLS IN APOPTOSIS In general, sulfhydryl groups are highly reactive and can participate in several different types of reactions. These groups also have a particular role in cellular defense against oxidative stress. Several studies indicate depletion of intracellular SH during apoptosis (6,8,16). Addition of NAC, a thiol antioxidant, can prevent factor-deprived cells from undergoing apoptosis (2,5) and apoptosis induced by TNF (17). Although a cellular decrease in glutathione is reported in association with apoptosis, it seems that other SH groups are implicated in apoptotic signaling since specific inhibition of glutathione biosynthesis with buthionine sulfoxamine did not induce apoptosis (16). Thioredoxin (Trx), another thiol-related antioxidant, has important roles in mediating apoptosis. Trx is a ubiquitous protein that contains two active cysteine residues in its catalytic site. Trx cooperates with methionine sulfoxide reductase to reactivate proteins damaged by previous oxidation of their methionine residues (11). Oxidation of active cysteines in Trx and subsequent loss of methionine sulfoxide activity can explain the promotion of calcium influx by the specific SH oxidant such as diazenedicarboxilic acid bis(N,N-dimethylamide) (17). Trx is reported to inhibit TNF- and Fas-mediated apoptosis (7). Trx is also reported to inhibit NFkB and to activate AP1 (18,19). NFkB and activator protein-1 (AP-1), two immunologically important transcription factors, have been found to be activated by redox-dependent processes. NFkB is activated by H2O2 (19). NFkB activation is not dependent on its de novo synthesis. In unstimulated cells, NFkB exists in the cytoplasm as an inactive complex bound to the inhibitory subunit 1kB. To be functional, NFkB must dissociate from its inhibitory subunit, 1kB, which must be phosphor-

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ylated with a protein kinase that has not yet been identified and then degraded by a protease (20). This unidentified kinase is supposed to be activated by oxidative modification of a critical cysteine residue (21) in its molecule or in other related regulating proteins. Oxidation of intracellular SH can also activate protein tyrosine kinases (22). Protein tyrosine kinases are reported to be involved in Fas-mediated apoptosis (23). Perhaps in this way, NFkB activation may represent an essential step in apoptosis. Baeuerle et al. (24) hypothesized, on the basis of a relation between free radical production and NFkB activation and the inhibition of NFkB activation by NAC, that ROS are second messengers for signal transduction of inflammatory signals. SOD AND Bcl-2 IN APOPTOSIS SOD is responsible for the transformation of the highly reactive and toxic O20 . Electron transfer chains located in the mitochondrion, endoplasmic reticulum, and nucleus are major intracellular sources of O20 . Intracellularly, there are two forms of SOD, MnSOD in mitochondria and CuZnSOD in cytoplasm. H2O2 resulting from SOD activity is transformed into H2O and O2 by two enzymes: catalase and glutathione peroxidase. The apoptosis induced by the rapid rise in the intracellular ROS due to TNF receptor stimulation is correlated with the SOD level. Troy and Shelanski (1994) found that a decrease in SOD in PC12 cells to õ40% of constitutive levels results in rapid cell death by apoptosis. Inhibition of apoptosis by vitamin E is mediated by free radicals and not by some other effect of down-regulating SOD (25). The protein encoded by Bcl-2 is also responsible for defense against oxidative stress. This is a 26-kDa protein associated with mitochondrial membranes, endoplasmic reticulum, and the nuclear envelope, sites for free radical generation (26). Bcl-2 is a mammalian homologue of ced 9 in nematodes and is important for cell survival. The location of bcl-2 in cell membranes is not essential for its function, since proteins missing the anchor segment still possess the ability to be antioxidants (2). However, this may not be the sole function of Bcl-2 since it is found to protect the cell even under anaerobic conditions (27). Antioxidants can substitute in vitro for Bcl-2 expression. However, Bcl-2 does not inhibit formation of free radicals, but blocks their damaging effects. Overexpression of Bcl-2 can inhibit lipid peroxida-

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tion (2). In vitro, Bcl-2 protein has no demonstrated peroxidase ability (28). Therefore it has been assumed that Bcl-2 acts as a free radical scavenger. Bcl-2-deficient mice showed apoptotic involution of the thymus and spleen, polycystic kidneys, and hair hipopigmentation (29,30). Hair hipopigmentation is directly correlated with free radical generation. Biosynthesis of melanin can generate free radicals. Melanocytes have low levels of SOD and peroxidase. The absence of Bcl-2 makes the cell vulnerable to free radical attack. Transgenic mice that overexpressed Bcl-2 develop high-grade lymphomas. Thus, absence of Bcl-2 determines thymus apoptotic involution, while its overexpression leads to high-grade lymphomas. If a primary role of Bcl-2 is as a free radical scavenger, this experiment (30) supports the hypothesis that apoptosis is mediated by free radicals. PROTEASES AND POLY(ADP-RIBOSE) POLYMERASE (PARP) IN APOPTOSIS The amounts of the antioxidative enzymes normally present in the tissue are only sufficient to cope with usual rates of O20 , H2O2 , and NO generation. If the quantities of these reactive species are higher than usual, the response of the cell is to enhance the activity of protective enzymes, to activate repair systems, and, if the damage cannot be fixed, to activate cell death. Repair systems consist of the activation of methionine sulfoxide reductase, previously discussed; DNA-repairing poly(ADP-ribose) polymerase (an enzyme that has NAD as substrate); and proteases that degrade abnormal proteins. Proteins damaged by reactive oxygen species are usually cleaved by specific proteases (11). The activity of these proteases seems to decay with age. Their relationship with apoptotic cell death has not yet been studied. However, the dysfunction of these proteases is associated with the formation of b-amyloid in Alzheimer’s disease, a disease associated with excessive neuronal apoptotic death (43). Reactive oxygen species can cause DNA strand breakage, resulting in activation of PARP. Activation of PARP may lead to NAD and ATP depletion, and, as a consequence, GSH depletion, as well as the inability to maintain low intracellular free calcium (31,32). PARP activation has been observed during apoptotic processes (30,33). When DNA damage cannot be repaired, a protease degrading PARP is probably activated. This protease belongs to the ICE/ CED3 family of related proteases, and the name proposed for it is apopain (30). Specific inhibition of

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apopain blocks apoptosis, suggesting its importance in this process. Proteolytic cleavage of PARP will activate specific endonuclease normally inhibited by PARP, leading to cell death. DISEASES AND APOPTOSIS If one of the steps in the regulation of apoptotic cell death functions improperly, the results can be dramatic: cells die inappropriately, too much, or not at all. Apoptosis appears to be the mechanism of cell death in a wide variety of diseases, and oxidative stress has been postulated to contribute to the pathogenesis of some of these diseases. Elevated production of free radicals and/or depletion of antioxidants has been observed in various pathologic situations such as AIDS, cancer, and neurological and inflammatory diseases. Roederer and colleagues (34) maintained that one of the more important aspects of AIDS is the chronic inflammatory and oxidative stress that accompanies the infection. Oxidative stress may contribute to the loss of CD4 (helper) T cells, and HIV-infected persons clearly have decreased levels of thiols in their blood (35,36). Since NAC, which acts both as a precursor of glutathione and as an antioxidant by itself, can replenish depleted glutathione levels in vivo, resulting in suppression of apoptosis induced by HIV infection, it was suggested that it could be used as an adjunct in the treatment of AIDS (37). Thus, sulfhydryl oxidation may mediate the apoptosis induced by HIV infection (38). Mutations in the CuZnSOD gene are associated with extensive apoptotic death of motor neurons due to a decrease in the ability of a cell to detoxify free radicals, leading to familial amyotrophic lateral sclerosis (ALS) (39,40). The finding that superoxide-induced death can be specifically inhibited by treatment with antioxidants like vitamin E or xanthine oxidase inhibitors suggests that this mechanism is mediated by free radicals. Two common disorders associated with cell death are myocardial infarction and stroke. These diseases arise primarily as a result of necrosis. However, outside the central ischemic zone, cells die over a longer period and morphologically appear to die by apoptosis. Known inhibitors of apoptosis in vitro have been shown to limit infarct size in these disorders. Further tissue injury frequently occurs during establishment of reperfusion. Reperfusion is associated with acute increases in free radical production and intracellular calcium levels, both potent inducers of apoptosis (41).

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The intensity of the original insult may determine the ensuing pathway to either necrotic or apoptotic death. Lennon et al. (42) showed that it was not the type of agent but the concentration and/or duration of application of the agent that was responsible for determining whether an apoptotic or a necrotic mode of cell death would occur. Peroxynitrite (OONO0), formed by interaction of 0 O2 with NO and N-methyl-D-aspartate can induce neurotoxicity at low levels, predominantly due to an apoptotic mechanism, and necrotic cell damage at high levels (3). Bcl-2, which blocks either the formation of oxygen radicals or their effect, seems to protect cells not only from apoptotic death, which may be triggered by modest amounts of oxygen radicals, but also from unprogrammed necrotic death that occurs under conditions that normally make oxygen radicals levels soar. CONCLUSIONS Free radicals may trigger apoptosis directly as a result of oxidative stress and/or may function as intermediate steps in destructive pathways triggered by other agents. Thus, apoptosis may be regulated by modulation of free radical levels. REFERENCES 1. Stuehr DJ, Nathan CF. Nitric oxide: A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169:1543–1555, 1989. 2. Hockenbery DM, Oltovoi ZN, Yin XM. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251, 1993. 3. Bonfoco E, Krainic D, Ankarcrona M, Nicotera P, Lipton SA. Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methyl-Daspartate or nitric oxide/superoxide in cortical cultures. Proc Natl Acad Sci USA 92:7162–7166, 1995. 4. Hanson M, Asea A, Ersson U, Hermodsson S, Hellstrand K. Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. J Immunol 156:42–47, 1996. 5. Tilly JL, Tilly KI. Inhibitors of oxidative stress mimic the ability of follicle-stimulating hormone to suppress apoptosis in cultured rat ovarian follicles. Endocrinology 136(1):242– 252, 1995. 6. Slater AFG, Nobel CSI, Maelaro E, Bustamante J, Kimland M, Orenius S. Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway in thymocyte apoptosis. Biochem J 306:771–778, 1995. 7. Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol Today 15(1):7–10, 1994. 8. Orenius S, McConkey DJ, Nicotera P. Mechanism of oxidant-

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