Oxidative Stress and Apoptosis

Pathophysiology 7 (27) 153–163

Review

Oxidative stress and apoptosis

Krishnaswamy Kannan a, Sushil K. Jain b,*a Department of Medicine, Centre of Excellence for Arthritis and Rheumatology, Lousiana State Uni6ersity Health Sciences Center, Shre6eport,

LA 71130-3932, USAb Department of Pediatrics, Lousiana State Uni6ersity Health Sciences Center, 1501 Kings Highway, Shre6eport, LA71130-3932, USA

Received 9 March 2000; accepted 15 May 2000

Abstract

Apoptosis or programmed cell death, is essential for the normal functioning and survival of most multi-cellular organisms. Themorphological and biochemical characteristics of apoptosis, however, are highly conserved during the evolution. It is currentlybelieved that apoptosis can be divided into at least three functionally distinct phases, i.e. induction, effector and execution phase.Recent studies have demonstrated that reactive oxygen species (ROS) and the resulting oxidative stress play a pivotal role inapoptosis. Antioxidants and thiol reductants, such as N-acetylcysteine, and overexpression of manganese superoxide (MnSOD)can block or delay apoptosis. Bcl-2, an endogenously produced protein, has been shown to prevent cells from dying of apoptosisapparently by an antioxidative mechanism. Taken together ROS, and the resulting cellular redox change, can be part of signaltransduction pathway during apoptosis. It is now established that mitochondria play a prominent role in apoptosis. Duringmitochondrial dysfunction, several essential players of apoptosis, including pro-caspases, cytochrome C, apoptosis-inducing factor(AIF), and apoptotic protease-activating factor-1 (APAF-1) are released into the cytosol. The multimeric complex formation ofcytochrome C, APAF-1 and caspase 9 activates downstream caspases leading to apoptotic cell death. All the three functionalphases of apoptosis are under the influence of regulatory controls. Thus, increasing evidences provide support that oxidative stressand apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases

including AIDS, autoimmunity, cancer, diabetes mellitus, Alzheimer’s and Parkinson’s and ischemia of heart and brain. © 2000Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Reactive oxygen species; Apoptosis-inducing factor; Apoptotic protease-activating factor-1

www.elsevier.com/locate/pathophys

1. Introduction

Apoptosis, or programmed cell death (PCD), is anaturally occurring cell death process, essential for thenormal development and homeostasis of all multicellu-lar organisms [1]. This process is also important forremoving damaged, infected, or potentially neoplasticcells. However, both too little and too much apoptoticcell death can lead to adverse biological consequences[24]. Rheumatoid arthritis and cancer are the best ex-amples for too little apoptosis. Ischemic heart disease,AIDS and neurodegenerative diseases such as

Alzheimer’s, and Parkinson’s are the best examples oftoo much apoptosis [2–6]. The commercial availabilityof an ever-growing list of reagents, an explosive numberof research publications, and a number of scientificmeetings devoted to apoptosis signifies the relative im-portance of this emerging field [6].

Apoptosis is a cell death process that is clearly dis-tinct from necrosis. The integrity of the cell membraneis severely compromised or damaged in necrosis, lead-ing to cell swelling and cell lysis. Often, necrosis occursin a group of cells, or in tissue at a particular locus. Incontrast, apoptosis occurs at the single cell level. Dur-ing this process, an individual cell undergoes an activeprocess of cell death, set in motion by a genetic pro-gram and culminating in DNA fragmentation and theformation of membrane-packaged bits called apoptotic

* Corresponding author. Tel.: +1-318-6756086; fax: +1-318-6756059.

E-mail address: [email protected] (S.K. Jain).

0928-4680/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.PII: S 0 9 2 8 -4680 (00 )00053 -5

K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153–163154

bodies. The plasma membrane of early apoptotic cellsand the apoptotic bodies maintain their integrity, pre-venting leakage of cellular material and thereby aninflammatory response and local tissue damage. Bio-chemical changes on the outer surface of plasma mem-brane are recognized by tissue macrophages and severalother neighboring cell types. Apoptotic cells are effi-ciently cleared in vivo by antigen processing cells within2–4 h [1,3,5].

Apoptosis can be triggered by numerous factors in-cluding receptor-mediated signals, withdrawal ofgrowth factors, anti-tumor drugs and, under certainconditions, damage to DNA (1–7). A partial list ofsome of these inducers is included in Table 1. Each ofthese stimuli has its own specific pathway that leads toactivation of apoptotic process, however, all appear toconverge at a highly conserved sequence of events. Thecaspases are central component of this apoptotic pro-gram [8]. While the initial signal for apoptotic program-ming may vary, the morphological and biochemicalcharacteristics of PCD are uniformly similar, and are

highly conserved during the evolution of species. Theseobservations raise the intriguing possibility of the co-existence of multiple signaling pathways that convergeupstream of a common mechanism of events, predis-posing the cell to apoptosis [1–5]. It is currently be-lieved that one such convergent scheme involves theactive participation of mitochondria [9–12].

It is now well established that mitochondria is themain site of the generation of oxygen radicals, such as,superoxide anion, hydroxyl radical, singlet oxygen andhydrogen peroxide [7,13]. It is estimated that 1–4% ofoxygen reacting with the respiratory chain leads to theformation of superoxide radicals (O2

�−). Other sourcesof reactive oxygen species include radiation, cytotoxicchemicals and drugs. The formation and the sources offree radicals have been the subject of many reviews[9,14–17].

Excess oxidative stress kills cells either by necrosis orby apoptosis [9,10]. In many models of apoptosis, alter-ations in the redox status of the cell to a more oxidizingenvironment occurs prior to activation of the finalphase of caspase activation [10–15]. This argument isfurther supported by the ability of various anti-oxidantssuch as N-acetylcysteine (NAC) to block apoptosis in asimilar way that caspase inhibitors do [18]. The anti-ox-idant properties of Bcl-2, a potent inhibitor of apopto-sis, further supports this view [19–22]. Under normalconditions, aerobic cells are endowed with extensiveanti-oxidant defense mechanisms to counteract thedamaging effects of ROS [7,13,14]. When pro-oxidantsoverwhelm anti-oxidant defense mechanisms, oxidativestress occurs. Interestingly, apoptosis may serve as afail-safe device to prevent cells from running amok andproliferating uncontrollably in the face of a persistentoxidative stress [23]. This review presents an overviewof the relationship between oxidative stress and apopto-sis and its relevance to human health.

2. Mediators of apoptosis

Most of the mediators of apoptosis can be broadlyplaced in one of five categories based on the primaryperturbation within the cell, i.e. (1) the cell surface, (2)the cytosol, (3) the cytoskeleton, (4) the mitochondrion,(5) and the nucleus. At the cell surface, some of thewell-characterized death receptors are Fas (CD95), tu-mor necrosis factor receptor-1 (TNF-Rl), CAR1, DR3,DR4, and DR5 [24–29]. The ligands that activate thesedeath receptors are structurally related molecules thatbelong to the tumor necrosis factor (TNF) gene super-family [1,30]. Interaction of death receptors with lig-ands may lead to the initiation of a death signal,depending upon the presence of intracellular deathdomains and its physical association with the adapterprotein called Fas-associated death domain (FADD)

Table 1A partial list of inducers of oxidative stress and or apoptosis

Inducers of oxidative stressReactive oxygen Superoxide radical, hydroxyl

species radial, hydrogen peroxideIron, cadmium, mercuricMetalschloride

Pathophysiologic Hyperglycemia, ischemic heartdisease, Alzheimer’s andconditionParkinson’s

Inducers of apoptosis that in most cases, involve severe oxidativestress

H2O2, diamide, etoposide andPro-oxidantssemiquinones

Ionizing radiation Gamma UV radiationProtein synthesis inhibitor CycloheximideApoptotic stimuli Fas (CD95), TNFa, ceramide,

glutamate, growth factorwithdrawl (IL-2, IL-3, nervegrowth factor and serumstarvation)

Physiologic stimuli Glucocorticosteroids, calcium,TNF-a, glutamate

Pathophysiologic Serum starvation, IL-2 andconditions IL-3 withdrawal,

hyperglycemia, ischemia andreperfusion

Pro-apoptotic genes p53, Bax, c-mycOrganic solvents and Benzene and their metabolites

and 2,5-hexanedionemetabolitesDDT, endosulfan, dieldrin,Pesticidesand 2,3,7,8tetrachrolorodibenzo-p-dioxin

Drugs Actinomycin D, cisplatin,cycloheximide, taxol,camptothecin andstaurosporine

K. Kannan, S.K. Jain / Pathophysiology 7 (2000) 153–163 155

[28,29]. Additionally, FADD also contains anotherdeath effector domain that communicates with caspase8, one of the key enzymes in the death machinery. This,in turn, activates downstream caspases, thus commit-ting the cell to apoptosis. Further examples of ligandsthat act at the cell surface include the CD40/CD40ligand on T and B cells, and the glutamate/glutamatereceptor in the nervous system [28,31–33].

A major shift in the focus of research on apoptosisfrom the nucleus to the cytoplasm has occurred inrecent years. A substantial fraction of the pro-apoptoticmembers is localized in the cytosol or cytoskeleton[12,13,30]. Following a death signal, the pro-apoptoticmembers undergo a conformational change that en-ables them to target and integrate various subcellularsystems including mitochondrial outer membrane [34].Another major player in cell death by apoptosis is aclass of enzymes called ‘caspases’, which are cysteine-dependent enzymes and are sensitive to the redox statusof the cell [8,30,35,36]. These cysteine proteases aresynthesized as precursors that have little, if any, cata-lytic activity. The precursor is usually converted to theactive protease by proteolytic processing. One role ofcaspases is to inactivate proteins that are vital to cellsurvival. These enzymes cut off the cell–cell contact tothe surrounding cells, disorganize the cytoskeleton, shutdown DNA replication and repair, interrupt splicing,destroy DNA, disrupt the nuclear structure, inducebiochemical changes on the cell surface for easy recog-nition by phagocytes, and finally disintegrate the cell tovesicular bodies [35,37–42]. At present, 13 differentcaspases have been identified and more are being addedto the list [37]. Initiation of apoptotic events, however,depends on the ability of the signaling complexes togenerate an active protease [8].

Poly (ADP-ribose) polymerase (PARP) is a cellularsubstrate for caspase 3 and 7. Caspase 3 has also beendemonstrated to cleave all of the three following sub-strates, DNA dependent protein kinase (DNA-PKcs,the 70 kDa protein component of the U1-ribonucle-oprotein (U1-70 kDa), and PARP, at very similar sites,defining the DXXD motif as the key determinant forcleavage specificity. Specific inactivation of these sub-strates initiate an irreversible stress on the organizationof nuclear DNA structure and the regulation of geneexpression [45,46,51].

Under normal conditions, mitochondria possess anefficient biochemical defense mechanism to neutralizethe effect mediated by ROS. This system is composedof GSH, glutathione peroxidase, glutathione reductase,superoxide dismutase, NADP dehydrogenase(NADPH), Vitamin E and C [13,18,43–46]. For exam-ple, superoxide radicals are scavenged by superoxidedismutase, leading to the production of hydrogen per-oxide, which again is detoxified by glutathione perox-idase or catalase. Oxidative stress can occur under

conditions when oxygen radicals production is greaterthan the detoxification capacity of the cell [47,48].Several lines of experimental evidence recognize themitochondrial dysfunction as one of the importantmediators of apoptosis [9–15,23,49–56].

3. Oxidative stress and its significance in biology

ROS has been shown to play both beneficial as wellas deleterious roles. At very low concentration, it mayact as a second messenger in some of the signal trans-duction pathways [57]. However, when produced inexcess, it can cause oxidative damage to many vitalcomponents of the cell. There exists a dynamic relation-ship between ROS production and antioxidant capacityof the given cell system. Some oxidation processes suchas cysteine oxidation play a role in a dynamic regula-tory process within the cell. Such a variation may causea drastic modulation of the oxidized or reduced ratio ofsignaling proteins, such as, transcription factors. Onemechanism through which these effectors may elicitoxidative stress is the small G-protein ‘Ras’. Indeed,Ras is suspected to activate a cascade of kinases viaROS production [58]. Similarly, transcriptional factorssuch as NF-kB, p53 and AP-1 have been shown to bemodulated by oxygen species (reviewed in [13]). Sub-lethal ROS production, therefore, can interfere withsignal transduction pathways. ROS, in particular H2O2,are indeed second messengers for various physiologicalstimuli, such as, angiotensin inflammatory cytokinesand growth factors or transforming factors [59,60].There is a paradox surrounding the physiological andpatho-physiological roles played by reactive oxygenspecies such as the superoxide radical. It is universallyaccepted that the production of superoxide radical byactivated neutrophils and other phagocytes is an essen-tial component of immunological defense mechanismsagainst bacteria [61].

Oxidative stress occurs when redox homeostasiswithin the cell is altered. It has been suggested that theglobal shutdown of mitochondrial function under con-ditions of oxidative stress could contribute to apoptosisbecause of the dramatic decrease in cellular energysupply. Injury to cells occurs only when the ROSoverwhelm the biochemical defenses of the cell [43,62–65]. Reactive oxygen species, (in particular, hydroxyradicals) can react with all biological macromolecules,i.e. lipids, proteins, nucleic acids and carbohydrates.The initial reaction generates a second radical, whichreacts with a second macromolecule and so on in acontinuing chain reaction. Among the more susceptibletargets are polyunsaturated fatty acids [48,63–65].

The polyunsaturated fatty acyl side chains, becauseof their susceptibility to oxidative damage in membranephopholipids, pose a constant threat to cellular in-


Table 2Diseases that are influenced by oxidative stress or apoptosis

Neurodegenerative diseases (Alzheimer’s, Parkinson’s)Autoimmune disease (e.g. rheumatoid arthritis)Human immunodeficiency virus (AIDS)Diabetes mellitusCancers of lung, colon, breast and othersAlcohol induced liver diseaseHepatitis-C induced liver diseaseIschemic reperfusion damage-heart, liver

promoting genetic changes that favor the origin oftumor cells of malignant type [78]. Interestingly, currentchemotherapeutic agents such as anthracycline-deriva-tives, which are frequently used as chemotherapeutics inthe treatment of numerous types of cancers, targetsome of these apoptotic pathways. For example, adri-amycin is known to chelate iron and generate ROS thatresult in apoptosis of cancer cells [1–3,80]. As similar tocancer, oxidative stress has also been implicated inAIDS disease [81]. In particular, the ‘bystander effect’in uninfected lymphocytes, in HIV infection, seems toinvolve increased apoptosis. This increased cell deathhas been linked to the effects of free extracellular gp120and Tat protein that may have a major impact on theprogression of AIDS [81,82].

Although several risk factors can trigger the develop-ment of insulin-dependent diabetes (IDDM), severalstudies suggest a role for ROS in beta-cell death anddisease progression [83,84]. In a pancreatic beta-cellline, stable transfectants of aldose reductase gene in-duced apoptosis by causing a redox imbalance [85].Taken together, these observations show that oxidativestress can negatively modulate the expression of genesthat control carbohydrate metabolism by repressing theinsulin signaling pathway. Accumulating evidence indi-cates that increased antioxidant defense systems andingestion of antioxidants reduce the susceptibility toIDDM in animal models or in human study [84][86,87]. In vitro studies have demonstrated inhibition ofapoptosis by the antisense nucleotide to the p65NF-kB[88]. Whether the intervention with antisense therapycan prevent apoptosis and the progression of the dis-ease, it needs further investigation. Gene therapy, there-fore, remains a big challenge in translating some of therecent advances made in molecular biology to patientcare.

Ischemia and reperfusion injury are other manifesta-tions of ROS-mediated injury during surgery [89]. Is-chemic injury occurs when the blood supply to an areaof tissue is cut off under physiological conditions, orduring surgery when blood vessels are cross-clamped,and in organs for transplant. Oxygen deprivation dur-ing this period leads to necrotic lesions depending onduration. On the other hand, restoration of bloodsupply leads to reperfusion injury, which is due tosudden increase of ROS in the target tissue or organ.Under the in vivo conditions, a complex interplaybetween endothelial cells, neutrophils and other cells ofimmune system occurs after anoxia-reoxygenation.Some of the consequences of this interaction are theoxidative cell damage including lipid peroxidation, acti-vation of inflammatory cytokines, followed by neu-trophil and macrophage attack on the reperfused tissue.A second consequence being accelerated apoptotic celldeath in these tissues resulting in various clinical com-plications [90]. Oxidative stress can also repress the

tegrity and function [62–72]. The lipid radicals gener-ated during the early encounter with an oxidant addmolecular oxygen to produce lipid dioxyl radical. Thispro-oxidant abstracts, an allylic hydrogen from anotherunsaturated side chain, producing a lipid hydroperoxide(LOOH) and thus propagating the chain [62–64]. Iron,a transition metal, is also well known for its crucial rolein the initiation of new lipid-radical chain reactions[65].

Lipid peroxidation is an important biological conse-quence of oxidative cellular damage and aging [66–73].A partial list of conditions or diseases that are likely toinvolve oxidative stress is given in Table 2. This in-cludes several neurodegenerative diseases and AIDS.Other sources of ROS include radiation (e.g. UV), toxicchemicals (e.g. paraquat and endosulfan) andchemotherapeutic agents (e.g. adriamycin andbleomycin) [13,74,75]. The classical view of ROS asvillains that indiscriminately destroy biological macro-molecules has undergone a shift, in which, positivephysiological roles are considered as well. In summary,oxidative stress can have positive responses, such as,proliferation or activation, as well as negative re-sponses, such as, lipid peroxidation, DNA damage, cellgrowth inhibition or cell death.

4. Redox imbalance and apoptosis

Oxidative stress has been implicated in the pathogen-esis of several disease processes including, ischemia/reperfusion injury, Alzheimer’s, Parkinson’s anddiabetes mellitus. In cancer, ROS has been implicatedin damage to DNA resulting in altered gene expression.Consequently, changes in cell cycle-related protein ex-pression, activation of proto-oncogenes, and the inacti-vation of some tumor suppressor genes have beenreported [76–78]. Yet another modulation that deservesmention here is the increased expression of Bcl-2protein that favors prolonged cell survival. Recently, atherapeutic approach led to the yet incomplete investi-gation of antisense Bcl-2 oligonucleotides in the treat-ment of B-cell lymphomas [79].

In colon cancer, it is believed that a mutated p53gene allows pre-cancerous cells to accumulate tumor-


activity of T-lymphocytes, and thus alter the immuneresponse. Depletion of intracellular GSH pool elicitsoxidative stress by raising the intracellular redox poten-tial, and causes a marked inhibition of the T cell-medi-ated immune response. In particular, IL-2 expression,an important cytokine, is decreased by oxidative stress.This could contribute to the alteration of protectiveimmune response as in patients suffering from rheuma-toid arthritis and HIV-infected people [13,91].

5. Genes, oxidative stress and apoptosis

Gene expression, in general, is modulated by bothphysiological and environmental stimuli. It appears thatoxidative stress acts as a pleiotropic modulator in boththese pathways. Indeed, ROS have been described assecond messengers for several growth factors and cy-tokines. It has been shown that the transcription factorssuch as NF-kB and AP-1, which are stimulated byROS, could mediate such inductions [13]. Most of theliterature has historically focused on gene induction andless on gene repression by ROS. Under conditions ofmild oxidative stress, cell cycle-related genes are re-pressed to increase the lengthening of G1-phase. A cellcycle arrest is required in order to assess the amount ofmacromolecule alterations, and, if necessary, to enterthe apoptotic pathway instead of carrying on the cellu-lar division process. This delay in cell cycle is impor-tant, since a base alteration could be converted into anirreversible mutation if a mismatch escaped the repairsystems before replication. Hence, there is a necessitynot to activate S-phase too quickly when a cellularstress occurs.

As compared with genomic DNA, mitochondrialgenome appears to be more sensitive to oxidative dam-age. The mitochondrion possesses its own genome andproduces its own RNAs that are necessary to its func-tion. Crawford et al., in 1998 [92] have shown thatmitochondrial RNAs undergo specific degradationupon oxidative stress. Similarly, a direct relationshipbetween glutathione oxidation and mtDNA damage inapoptosis has been suggested by Esteve and co-workers[93]. Mitochondrial DNA is also heavily damaged byROS at the bases, as indicated by the high steady-statelevel of 8-hydroxydeoxyguanosine, i.e. the presence ofwhich causes mispairing and point mutations [94].These mutations can be detected by long-extensionPCR, a method for detecting a variety of mutations ofmitochondrial genome [95]. In mammalian cells, UVBradiation has been reported to repress mitochondrialfunction by strongly inhibiting its transcription [96].Following the treatment of hamster fibroblasts withH2O2, these authors observed that the 16S rRNAs, amajor component of mitochondrial ribosomes, werespecifically degraded, whereas cytosolic mRNAs were

not. This resulted in a dramatic shutdown of mitochon-drial protein biosynthesis. Transcription factors such asNF-kB, p53 and AP-1 are also sensitive to redoxchanges in mammalian cells, primarily through redoxregulation of their DNA binding regions [13,84,88,97].

Cell death in the nematode Caenorhabditis elegans isundoubtedly the best system for the study the geneticsof programmed cell death. During development, a pro-cess resembling apoptosis deletes 131 cells out of 1090cells of this nematode. A total of 14 suicide genes thatare important for development have been identified sofar, of which three genes — Ced3, Ced4 and Ced9 —have received most attention. The Ced9 gene negativelyregulates the Ced3 and Ced4 genes [1,35,97–99]. Thehomologues of Ced9 in humans have been identified asBcl-2, which protect mammalian cells. Caspase 3(YAMA/CPP32), a proteolytic enzyme in humans isbelieved to be an equivalent of Ced3 in nematode. Thehomologue to Ced4 in human has been identified to beAPAF-1 (apoptotic protease-activating factor-1) [35].Other genes of importance are c-fos, c-jun as well asc-myc, p53, clusterin, RP-2 and RP-8 [100,101]. How-ever, the complex mechanisms controlling gene expres-sion that determines cell survival and cell death are stillunresolved.

6. Bcl-2 family members and apoptosis

Bcl-2 is the first mammalian regulator gene that wasidentified to have anti-apoptotic potential in a varietyof cell systems. At least 15 Bcl-2 family members havebeen identified in mammalian cells [107]. All memberspossess at least one of four conserved motifs known asBcl-2 homology domains (BH1–BH4). Generallyspeaking, family members that act as inhibitors of celldeath harbor at least three domains (BH1, BH2, andBH3), which are important for protein–protein interac-tion and the suppression of apoptosis, whereas BH3serves as the minimal ‘death domain’ in the prop-apop-totic members studied so far [11]. Some of the familymembers, such as Bax, Bak, and Bok, work in amanner opposite to that of Bcl-2, though they resembleBcl-2 closely. Pro- and anti-apoptotic family membersof Bcl-2 can homodimerize or heterodimerize and seem-ingly can neutralize one another’s function, suggestingthat their relative concentration in the given cell mayact as a rheostat for the suicide program. Bcl-2 islocalized on the cytoplasmic face of the mitochondrialouter membrane, endoplasmic reticulum and nuclearenvelope. It is currently believed that this strategiclocalization allows it to register and counterbalance theoxidative damage done to these compartments andaffect their behavior [19,108,109]. This pro-survivalprotein also seems to maintain the membrane integrityof mitochondria, by directly or indirectly preventing the


release of cytochrome C, which, along with dATP andAPAF-1, facilitates the activation of pro-caspase 9 toits active form of caspase 9 [11,108,109]. Bcl-2 protectscells against diverse cytotoxic insults, for example,gamma radiation, cytokine withdrawal, hypoxia, ROS,dexamethasone, staurosporine, and cytotoxic drugs. Allpro-survival Bcl-2-like genes are potentially oncogenic,whereas pro-apoptotic family members may act as tu-mor suppressors. Clarifying how Bcl-2 family membersgovern apoptosis under various conditions mightprovide clues for the development of novel therapies inclinical settings.

7. Oxidative stress and mitochondria

The mitochondria is sensitive to changes in the redoxstate of the cell. Several studies have shown that theglobal shutdown of mitochondrial function under con-ditions of oxidative stress could contribute to apoptosis[9–13]. Maintenance of mitochondrial membrane in-tegrity is a dynamic process. Under severe oxidativestress, the mitochondrial permeability transition (PT)occurs. PT involves a sudden increase of the innermitochondrial membrane permeability to solutesgreater than 1500 Da (protons, calcium, GSH etc.). It iscurrently believed that PT functions as a voltage sensor,a thiol sensor, a sensor of oxidation–reduction equi-librium of adenine nucleotide pool, and as a sensor ofdivalent cations. As a consequence, defective PT poreopening to larger molecules causes uncoupling of therespiratory chain resulting in hypergeneration of ROS,cessation of ATP synthesis, matrix Ca++ outflow anddepletion of reduced glutathione and other reductants.Following the inner membrane permeability and therelease of matrix solutes, a colloidal osmotic pressurearises in the mitochondrial matrix due to the highconcentrations of proteins, which are slow to equili-brate [101–103]. In order to correct the osmotic bal-ance, the diffusion of H2O results in a massive swellingof the mitochondria [102]. This change in membranepotential predisposes these cells to oxidative damage byimpairment of endogenous antioxidant defense mecha-nisms [49–56]. Another crucial step in the early changesin mitochondria is alteration of mitochondrialtransmembrane potential (DCm) [9–13]. A decline inthe DCm alone can induce oxidative stress and celldeath [12]. The measurement of cell respiration, per-formed with whole cell populations, showed that thelower DCm correlates, as expected, with an uncouplingof electron transport from ATP production [104,105].These events are detected at early stages of the apop-totic process before most of the cells are irreversiblycommitted to death suggesting that mitochondria couldbe a primary target during apoptosis. Recently, the

release of cytochrome C from mitochondria has beenshown as another important effector molecule in themediation of programmed cell death [12,56,106]. It ispostulated that cytochrome C is somehow able to inter-act with pre-existing cytoplasmic factor(s), which subse-quently mediate the cleavage of zymogens and resultingin the activation of caspase 9.

Permeability transition is also associated with therelease of apoptotic inducing factor (AIF), which hasrecently been identified and characterized [11,53,54].This is a �50 kDa protein located in the inner mito-chondrial membrane whose functions are inhibited byBcl-2. Under in vitro conditions, purified AIF inducesdose and temperature-dependent apoptosis in isolatednuclei. This is an important finding in that membranepermeability transition predisposes the mitochondria tolose protein thiols and other factors including AIF tothe cytosol for the initiation of a biochemical cascaderesulting in programmed cell death [11]. It is not clearat this time whether the release of AIF and cytochromeC is interrelated, or occurs as biochemical events ofseparate pathways. In both instances, Bcl-2, an anti-apoptotic protein, prevented the actions of AIF andcytochrome C release. This partly explains the impor-tance of the physical location of Bcl-2 on the outermembrane of the mitochondria, nuclear envelope andendoplasmic reticulum [53,54,110]. A schematic repre-sentation of the association between oxidative stressand apoptosis is presented in Fig. 1.

Calcium is another important mediator of apoptosis[111–113]. Also, a close relationship exists betweencalcium status and oxidative stress in many isolated cellsystems, the best example being isolated rat hepatocytes[114]. When cells are deprived of calcium, mitochondriabecome sensitive to this calcium-deficient state and theircalcium regulatory mechanism is disturbed. Calciumomission results in a marked decline of mitochondrialtransmembrane potential (DCm), leading to oxidativestress and induction of programmed cell death. Manyof the antioxidants can prevent this collapse, thus indi-cating that the DCm is critical to the process and isreversible in the early phases. However, it remains to bedetermined what dictates the efflux of mitochondrialcalcium. It must be emphasized here that oxidativestress induced by the absence of extracellular calcium isdistinct from that generated by ROS, in that it appearsto induce calcium cycling in and out of mitochondrialmatrix, which serves to promote oxidative stress[15,64,115]. In the human erythrocyte model, pretreat-ment of erythrocytes with calcium increases their sensi-tivity to calcium [115]. Thus, it appears that calciumcan promote lipid peroxidation both under conditionsof excess and of deficiency. Calcium homeostasis is,thus, vital to the function and survival of the cell [116].


8. Mitochondria and apoptosis

While the central role of mitochondria in apoptosishas been widely accepted, experimental evidence nowpoints to the existence of yet another pathway indepen-dent of mitochondria. However, it appears that boththese pathways involve pro-caspase 8 as one of theimportant intermediary step. Using the Xenopus cell-free system as the experimental model, Kuwana et al.,

in 1998 [117] have provided experimental evidence thatsuggests that caspase 8 participates in both pathways.In the absence of mitochondria, activation of a caspasecascade by caspase 8 produces only a partial apoptoticphenotype in nuclei added to the Xenopus cell extract.In contrast, the mitochondria-independent pathway,which involves the release of cytochrome C from mito-chondria into cytosol, triggers full nuclear apoptosis.Moreover, engagement of the mitochondria provides an

Fig. 1. Schematic model of mammalian cell death pathway. Death signal may be delivered at the cell surface by direct ligand-receptor interaction.This followed by clustering of death receptors and activation of caspase-8. Alternatively, cytotoxic drugs, ionizing radiation may directly activatecaspase-9, otherwise a later event. Mitochondria plays a central role in apoptosis by releasing apoptogenic factors and proteases into the cytosol.A major checkpoint in this pathway is the ratio between pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) members. Miitochondrial dysfunctionincludes PT, change in mitochondrial membrane potential, production of ROS, release of cytochrome C, AIPF-1 and caspases-2, -3, and -9 intothe cytosol, where they form multimeric complexes. This activates downstream caspases and degradation of death substrates in the nucleus, whichultimately leads to cell death.


efficient means of amplifying the apoptotic signal trans-duced by caspase 8 even under very low concentration.In the absence of mitochondria, high concentrations ofcaspase 8 were required to activate downstream caspasecascade. Interestingly, caspase 8 can activate the mito-chondria-dependent pathway even when the Bcl-2protein is present. This helps explain the failure of Bcl-2to inhibit CD95-dependent apoptosis consistently [117].Scaffidi et al., in 1998 [118] have identified two types ofCD95-mediated cell death. Recently, it has beendemonstrated that Apaf-1−/− and caspase 9−/− Tcells remain sensitive to Fas-induced killing [119,120].However, Fas-induced apoptosis was markedly reducedin embryonic fibroblasts with Apaf-1−/− phenotype[121], suggesting Fas (CD95) can activate differentpathways in different cell types. These observations inT cells indicate that the apoptotic function of mito-chondria (at least the cytochrome C release part) can bebypassed in these cells.

Other less defined pathways that lead to apoptosisare the ones initiated by potent cytotoxic chemicals orgamma irradiation. These pathways seem to rely on theapoptotic function of mitochondria, since cells withApaf-1−/− and caspase-9−/− phenotypes are princi-pally resistant to these death-inducing agents [119–121]. Further research in this area may help us to betterunderstand some of the missing links.

9. Conclusions

The role of oxidative stress as a mediator of apop-totic cell death in diverse cell systems is now betterunderstood. In recent years, mitochondria have gainedconsiderable importance both as a site for ROS produc-tion and as a major player in apoptosis. The involve-ment of mitochondria or ROS in apoptosis is notwithout controversy. In some experimental systems,apoptosis can occur at very low oxygen tension, whenROS are unlikely to occur. The protooncogeneproduct, Bcl-2, can inhibit apoptosis both in the pres-ence and in the absence of reactive oxygen products.Further research is needed to clarify the underlyingmechanisms.

Recent developments suggest that mtDNA may be agood indicator of oxidative damage in apoptosis. It isbecoming apparent that the redox status of a cell canhave a complex and multilayered effect on cell survivaland cell death. Future investigations will unravelwhether oxidative stress is a pre-requisite for all apop-totic events. This would be an attractive mechanismwhereby a panoply of seemingly diverse injuries couldrapidly converge on common apoptotic pathways.More in-depth studies are needed to elucidate the roleof BH3-domain-only subset of molecules and their in-terplay with ROS. This could open new sites of drug

targeting. Future studies may also improve our under-standing whether the oxidation states of mitochondrialthiols alone function as a prominent apoptotic sensor.A better understanding of the molecular machinery ofapoptosis in the pathophysiology will undoubtedlyprovide novel therapeutic interventions and care for alarge number of patients with chronic diseases.

Acknowledgements

We thank Georgia Morgn First for the critical read-ing of the manuscript. This work was supported in partby the funds by Feist Weiller Cancer Center and theCenter of Excellence for Arthritis and Rheumatology ofthe Louisiana State University Health Sciences Centerat Shreveport, and a grant-in aid from the AmericanHeart Association (Southeast).

References

[1] R.A. Schwartzman, L.A. Cidlowski, Apoptosis: the biochem-istry and molecular biology of programmed cell death, En-docrine Rev. 14 (1993) 133–151.

[2] C.B. Thompson, Apoptosis in the pathogenesis and treatmentof disease, Science 267 (1995) 1456–1462.

[3] L.F.R. Kerr, C.M. Winterford, B.N. Harmon, Apoptosis: itssignificance in cancer and cancer therapy, Cancer 73 (1994)2013–2026.

[4] R.C. Duke, D.M. Ojcius, D.E. Young, Cell suicide in healthand disease, Sci. Am. 275 (1996) 80–87.

[5] L.F.R. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basicbiological phenomenon with wide ranging implications in tissuekinetics, Br. J. Cancer 26 (1972) 239–257.

[6] Editorial: Cell Death in Us and Others. Science 281 (1998)1283.

[7] T.M. Burtke, P.A. Sandstrom, Oxidative stress as a mediator ofapoptosis, Immunol. Today 15 (1994) 7–10.

[8] G.S. Salvesen, V.M. Dixit, Caspase activation: the induced-proximity model, Proc. Natl. Acad. Sci. USA 96 (1999) 10964–10967.

[9] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A.Macho, T. Hirsch, S.A. Susin, P.X. Petit, B. Mignotte, G.Kroemer, Sequential reduction of mitochondrial transmem-brane potential and generation of reactive oxygen species inearly programmed cell death, J. Exp. Med. 182 (1995) 367–377.

[10] N. Zamzami, S.A. Susin, P. Marchetti, T. Hirsch, I. GomezMonterrey, M. Castedo, G. Kroemer, Mitochondrial control ofnuclear apoptosis, J. Exp. Med. 183 (1996) 1533–1544.

[11] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science281 (1988) 1309–1311.

[12] S.A. Susin, N. Zamzami, G. Kroemer, Mitochondria as regula-tors of apoptosis; doubt no more, Biochim. Biophys. Acta 1366(1998) 151–165.

[13] Y. Morel, Barouki. Repression of gene expression by oxidativestress, Biochem J (1999) 342: 481–496.

[14] A. Fernandez, J. Kiefer, L. Fosdick, D.J. McConkey, Oxygenradical production and thiol depletion are required for Ca2+-mediated endogenous endonuclease activation in apoptotic thy-mocytes, J. Immunol. 155 (1995) 5133–5139.


[15] C. Richter, Pro-oxidants and mitochondrial Ca2+ and theirrelationship to apoptosis and oncogenesis, FEBS 325 (1993)104–107.

[16] J.Z. Byczkowski, T. Gessner, Biological role of superoxide ionradical, Int. J. Biochem. 20 (1988) 569–580.

[17] I. Fridovich, Superoxide radical: an endogenous toxicant, Ann.Rev. Pharmacol. Toxicol. 23 (1983) 239–257.

[18] A.J. MeGowan, R.S. Fernandes, A. Samali, T.G. Cotter, Anti-oxidants and apoptosis, Biochem. Soc. Trans. 24 (1996) 229–267.

[19] D.M. Hockenberry, Bcl-2, a novel regulator of cell death,BioEssays 17 (1995) 631–638.

[20] S.J. Korsmeyer, X. Yin, Z.N. Oltvai, D.J. Veis-Novack, G.P.Linette, Reactive oxygen species and the regulation of cell deathby the Bcl-2 gene family, Biochim. Biophys. Acta 1271 (1995)63–66.

[21] D.M. Hockenbery, Z.N. Oltvai, X.M. Yin, C.L. Millman, S.J.Korsmeyer, Bcl-2 functions in an antioxidant pathway to pre-vent apoptosis, Immunol. Today 18 (1993) (1993) 44–51.

[22] D.W. Voehringer, Bcl-2 and glutathione: alterations in cellularredox state that regulate apoptosis sensitivity, Free Rad. Biol.Med. 27 (1999) 945–950.

[23] G. Kroemer, N. Zamzami, S.A. Susin, Mitochondrial controlof apoptosis, Immunol. Today 18 (1997) 44–51.

[24] S. Nagata, Fas-mediated apoptosis, Adv. Exp. Med. Biol. 406(1996) 119–124.

[25] H. Hsu, H.B. Shu, M.G. Pan, D.V. Goeddel, TRADD-TRAF2and TRADD-FADD interactions define two distinct TNF re-ceptor 1 signal transduction pathways, Cell 84 (1996) 299–308.

[26] R. Schwandner, K. Wiegmann, K. Bernardo, D. Kreder, M.Kronke, TNF receptor death domain-associated proteinsTRADD and FADD signal activation of acid sphingomyeli-nase, J. Biol. Chem. 273 (1998) 5916–5922.

[27] L. Kitson, T. Raven, Y.P. Jiang, D.V. Goeddel, K.M. Giles,K.T. Pun, C.J. Grinham, R. Brown, S.N. Farrow, A death-do-main-containing receptor that mediates apoptosis, Nature 384(1996) 372–375.

[28] A. Ashkenazi, V.M. Dixit, Death receptors: signaling and mod-ulation, Science 281 (1998) 1305–1308.

[29] P. Schneider, M. Thome, K. Burns, L.L. Bodmer, K. Hofmann,T. Kataoka, N. Holler, J. Tschopp, TRAIL receptors 1 (DR4)and 2 (DR5) signal FADD-dependent apoptosis and activateNF-kappa-B, Immunity 7 (1997) 831–836.

[30] I.E. Wertz, M.R. Hanley, Diverse molecular provocation ofprogrammed cell death, Trends Biochem. Sci. 21 (1996) 359–364.

[31] S. Lederman, A.M. Cleary, M.J. Yellin, D.M. Frank, M.Karpusas, D.W. Thomas, L. Chess, The central role of theCD40-ligand and CD40 pathway in T-lymphocyte-mediateddiffirentiation of B lymphocytes, Curr. Opin. Hematol. 3 (1996)77–86.

[32] G.G. Klaus, M.S. Choi, E.W. Lam, C. Johnson-Leger, J. Cliff,CD40: a pivotal receptor in the determination of life/deathdecisions in B lymphocytes, Int. Rev. Immunol. 15 (1997) 5–31.

[33] Y. Du, K.R. Bales, R.C. Dodel, E. Hamilton-Byrd, L.W. Horn,D.L. Czilli, L.K. Simmons, B. Ni, S.M. Paul, Activation of acaspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons,Proc. Natl. Acad. Sci. USA 94 (1997) 11657–11662.

[34] A. Gross, J.M. McDonnell, S.J. Korsmeyer, Bcl-2 family mem-bers and the mitochondria in apoptosis, Genes Develop. 13(1999) 1899–1911.

[35] N.A. Thornberry, Y. Lazebnik, Caspases: enemies within, Sci-ence 281 (1998) 1312–1316.

[36] M.B. Hampton, S. Orrenius, Redox regulation of apoptotic celldeath in the immune system, Toxicol. Lett. 102-103 (1998)355–358.

[37] E.S. Alnemri, D.J. Livingston, D.W. Nicholson, G. Salvesen,N.A. Thomberry, W.W. Wong, J. Yuan, Human ICE/CED-3protease nomenclature, Cell 87 (1996) 171.

[38] S.L. Martin, D.R. Green, Protease activation during apoptosis:death by a thousand cuts?, Cell 82 (1995) 349–352.

[39] B. Zhivotovsky, D.H. Burgess, D.M. Vanags, S. Orrenius,Involvement of proteolytic machinery in apoptosis, Biochem.Biophys. Res. Comm. 230 (1997) 481–488.

[40] L. Casciola-Rosen, D.W. Nicholson, T. Chong, K.R. Rowan,N.A. Thornberry, D.X. Miller, A. Rosen, Apopain/CPP32cleaves proteins that are essential cellular repair: a fundamentalprinciple of apoptotic death, J. Exp. Med. 183 (1996) 1957–1964.

[41] B. Zhivotovsky, A. Gahm, M. Ankarcrona, P. Nicotera, S.Orrenius, Multiple proteases are involved in thymocyte apopto-sis, Exp. Cell. Res. 221 (1995) 404–412.

[42] P.A. Henkart, ICE family proteases: mediators of all apoptoticcell death?, Immunity 4 (1996) 195–201.

[43] H. Sies, Oxidative Stress, Academic Press, London, 1985.[44] N. Sato, S. Iwata, K. Nakamura, T. Hori, K. Mori, J. Yodoi,

Thiol-mediated redox regulation of apoptosis. Possible roles ofcellular thiols other than glutathione in T cell apoptosis, J.Immunol. 154 (1995) 3194–3203.

[45] L.P. Kehrer, L.G. Lund, Cellular reducing equivalents andoxidative stress, Free Radic. Biol. Med. 17 (1994) 65–75.

[46] D.J. Reed, M.K. Savage, Influence of metabolic inhibitors onmitochondrial permeability transition and glutathione status,Biochim. Biophys. Acta 1271 (1995) 43–50.

[47] A. Kowaltowski, A.E. Vercesi, Mitochondrial damage inducedby conditions of oxidative stress, Free Radic. Biol. 26 (1999)463–471.

[48] B Halliwell, J.M.C. Gutteridge, Free Radicals in Biology andMedicine, Oxford University Press, Oxford, 1989.

[49] P.X. Petit, S.A. Susin, N. Zamzami, B. Mignotte, G. Kroemer,Mitochondria and programmed cell death: back to the future,FEBS Lett. 396 (1996) 7–13.

[50] P. Marchetti, M. Castedo, S.A. Susin, N. Zamzami, T. Hirsch,A. Macho, A. Haeffner, F. Hirsch, M. Geuskens, G. Kroemer,Mitochondrial permeability transition is a central coordinatingevent of apoptosis, J. Exp. Med. 184 (1996) 1155–1160.

[51] M. Castedo, A. Macho, N. Zamzami, T. Hirsch, P. Marchetti,J. Uriel, G. Kroemer, Mitochondrial perturbations definelymphocytes undergoing apoptotic depletion in vivo, Eur. J.Immunol. 25 (1995) 3277–3284.

[52] N. Zamzami, T. Hirsch, B. Dallaporta, P.X. Petit, G. Kroemer,Mitochondrial implication in accidental and programmed celldeath: apoptosis and necrosis, J. Bioenerg. Biomembr. 29(1997) 185–193.

[53] S.A. Susin, N. Zamzami, M. Castedo, T. Hirsch, P. Marchetti,A. Macho, E. Daugas, M. Geuskens, G. Kroemer, Bcl-2 in-hibits the mitochondrial release of an apoptogenic protease, J.Exp. Med. 184 (1996) 1331–1341.

[54] I. Marzo, S.A. Susin, P.X. Petit, L. Ravagnan, C. Brenner, N.Larochette, N. Zamzami, G. Kroemer, Caspases disrupt mito-chondrial membrane barrier function, FEBS Lett. 427 (1998)198–202.

[55] L.L. Scarlett, M.P. Murphy, Release of apoptogenic proteinsfrom the mitochondrial intermembrane space during the mito-chondrial permeability transition, FEBS Lett. 418 (1997) 282–286.

[56] J.C. Yang, G.A. Cortopassi, 1998. Induction of the mitochon-drial permeability transition causes release of the apoptogenicfactor cytochrome C, Free Radic. Biol. Med. 24 (1998) 624-631.

[57] Y.J. Suzuki, H.J. Forman, A. Sevanian, Oxidants as stimula-tors of signal transduction, Free Radic. Biol. Med. 22 (1997)269–285.


[58] E. Pennesi, Superoxide relay Ras protein’s oncogenic message,Science 275 (1997) 1567–1568.

[59] M. Ushio-Fukai, R.W. Alexander, M. Akers, K.K. Griendling,p38 Mitogen-activated protein kinase is a critical component ofthe redox-sensitive signaling pathways activated by angiotensinII. Role in vascular smooth muscle cell hypertrophy, J. Biol.Chem. 273 (1998) 15022–15029.

[60] Y.S. Bae, S.W. Kang, M.S. Seo, I.C. Bainers, E. Tekle, P.B.Chock, S.G. Rhee, Epidermal growth factor (EGF)-inducedgeneration of hydrogen peroxide. Role in EW receptormediatedtyrosine phosphorylation, J. Biol. Chem. 272 (1997) 217–221.

[61] B.M. Babier, Oxygen-dependent microbial killing by phago-cytes, New Engl. J. Med. 298 (1978) 659–668.

[62] A.A. Horton, S. Fairhurst, Lipid peroxidation and mechanismsof toxicity, CRC Crit. Rev. Toxicol. 18 (1987) 27–79.

[63] H. Jaescheke, Mechanisms of oxidant stress-induced acute tis-sue injury, Proc. Soc. Exp. Biol. Med 209 (1995) 104–111.

[64] D.J. Reed, Toxicity of oxygen, in: F. De Matteis, L.L Smith(Eds.), Molecular and Cellular Mechanisms of Toxicity, CRCPress, New York, 1995, pp. 35–68.

[65] H. Sies, Biochemistry of oxidant stress, Agrew. Chem. Int. Ed.25 (1986) 1058–1071.

[66] K. Kannan, S.K. Jain, Effect of high glucose on cellularproliferation and lipid peroxidation in cultured Vero cells,Horm. Metab. Res. 26 (1994) 322–325.

[67] S.K. Jain, The accumulation of Malonyldialdehyde, a productof fatty acid peroxidation, can disturb aminophospholipid orga-nization in the membrane bilayer of human erythrocytes, J.Biol. Chem. 25 (1984) 3391–3394.

[68] S.K. Jain, In vivo externalization of phosphatidylserine andphosphatidylethanolamine in the membrane bilayer and hyper-coagulability by the lipid peroxidation of erythrocytes in rats, JClin. Invest. 76 (1985) 281–286.

[69] S.K. Jain, The neonatal erythrocyte and its oxidative suscepti-bility, Semin. Hernatol. 26 (1989) 286–300.

[70] S.K. Jain, Evidence for membrane lipid peroxidation during thein vivo aging of human erytbrocytes, Biochim. Biophys. Acta937 (1988) 205–210.

[71] S.K. Jain, K. Kannan, G. Lim, Ketosis (acetoacetate) cangenerate oxygen radicals and cause increased lipid peroxidationand growth inhibition in human endothelial cells, Free Radic.Biol. Med. 25 (1998) 1083–1088.

[72] R.J. Reiter, J.M. Guerrero, J.J. Garcia, D. Acuna-Castroviejo,Reactive oxygen intermediates, molecular damage, and aging.Relation to melatonin, Ann. N.Y.Acad. Sci. 854 (1998) 410–424.

[73] Y. Christen, Oxidative stress and Alzhieimer disease, Am. J.Clin. Nutr. 71 (2000) 621S–629S.

[74] K. Kannan, R.F. Holcombe, S.K. Jain, R. Chervenak, R.E.Wolf, J. Glass, Evidence for the induction of apoptosis byendosulfan in a human T-cell leukemic line, Mol. Cell Biochem.205 (2000) 53–66.

[75] H. Kappus, Oxidative stress in chemical toxicity, Arch. Toxicol.60 (1987) 144–149.

[76] J.M. Mates, F.M. Sanchez-Jimenez, Role of reactive oxygenspecies in apoptosis: implications for cancer therapy, Int. J.Biochem. Cell Biol. 32 (2000) 157–170.

[77] S. Ambs, S.P. Hussain, A.J. Maarrogi, C.C. Harris, Cancer-prone oxyradical overload disease, IARC Sci. Publ. 150 (1999)295–302.

[78] J.M. Lee, A. Bernstein, Apoptosis, cancer and the p53 tumoursuppressor gene, Cancer Metastasis Rev. 14 (1995) 149–161.

[79] F.E. Cotter, Antisense therapy of hematologic malignancies,Semin. Hematol. 36 (1999) 9–14.

[80] I. Muller, D. Niethammer, G. Bruchelt, Anthracycline-derivedchemotherapeutics in apoptosis and free radical cytotoxicity,Int. J. Mol. Med. 1 (1998) 491–494.

[81] D. Romero-Alvira, E. Roche, The keys of oxidative stress inacquired immune deficiency syndrome apoptosis, Med. Hy-potheses 51 (1998) 169–173.

[82] S.R. Bartz, M. Emerman, Human immunodeficiency virus type1 Tat induces apoptosis and increases sensitivity to apoptoticsignals by up-regulating FLICE/caspase-8, J. Virol. 73 (1999)1956–1963.

[83] O. Guigliano, A. Ceriello, Oxidative stress and diabetic vascularcomplications, Diab. Care 19 (1996) 257–267.

[84] E. Ho, T.M. Bray, Antioxidants, NFkappaB activation, anddiabetogenesis, Proc. Soc. Exp. Biol. Med. 222 (1999) 205–213.

[85] R. Hamaoki, J. Fujii, J. Miyagawa, M. Takahashi, M. Kishi-moto, M. Moriwaki, K. Yamamoto, Y. Kajimoto, Y. Ya-masaki, T. Hanafusa, Y. Matsuzawa, N. Taniguchi,Overexpression of the aldose reductase gene induces apoptosisin pancreatic beta-cells by causing redox imbalance, J. Biochem.(Tokyo) 126 (1999) 41–47.

[86] R.A. Kowluru, R.L. Engerman, T.S. Kern, Diabetes-inducedmetabolic abnormalities in myocardium: effect of antioxidanttherapy, Free Radic. Res. 32 (2000) 67–74.

[87] H. Kaneto, Y. Kajimoto, J. Miyagawa, T. Matsuoka, Y. Fuji-tani, Y. Umayahara, T. Hanafusa, Y. Matsuzawa, Y. Ya-masaki, M. Hori, Beneficial effects of antioxidants in diabetes:possible protection of pancreatic b-cells against glucose toxicity,Diabetes 48 (1999) 2398–2406.

[88] X. Du, K. Stockklauser-Farber, P. Rosen, Generation of reac-tive oxygen intermediates, activation of NF-KB and inductionof aoptosis in human endothelial cells by glucose: role of nitricoxide synthase, Free Radic. Biol. Med. 27 (1999) 752–763.

[89] D.L. Carden, D.N. Granger, Pathophysiology of ischeamia-reperfusion injury, J. Pathol. 190 (2000) 255–266.

[90] G.L. Semenza, Cellular and molecular dissection of reperfusioninjury: ROS within and without, Circ. Res. 86 (2000) 117–118.

[91] A.C. WaJsh, S.G. Michaud, L.A. Malossi, D.A. Lawrence,Glutathione depletion in human T lymphocytes: analysis ofactivation-associated gene expression and the stress response,Toxicol. Appl. Pharmacol. 133 (1995) 249–261.

[92] D.R. Crawford, N.E. Abramova, K.J. Davies, Oxidative stresscauses a general, calcium-dependent degradation of mitochon-drial polynucleotides, Free Radic. Biol. Med. 25 (1998) 1106–1111.

[93] J.M. Esteve, J. Mompo, J. Garcia, J.G.D. Asuncion, J. Sastre,M. Asensi, J. Boix, J.R. Vina, F.V. Pallardo, Oxidative damageto mitochondrial DNA and glutathione oxidation in apoptosis:studies in vivo and in vitro, FASEB J. 13 (1999) 1055–1064.

[94] C. Richter, Reactive oxygen and DNA damage in mitochon-dria, Mutat. Res. 275 (1992) 249–255.

[95] D.C. Wallace, Mitochondrial diseases in man and disease,Science 283 (1999) (1999) 1482–1487.

[96] T.M. Vogt, L. Welsh, W. Stolz, E. Kullmann, B. Jung, N.Landthaler, M. McClelland, RNA fingerprinting displays UVB-specific disruption of transcriptional control in humanmelanocytes, Cancer Res. 57 (1997) 3554–3561.

[97] G.T. Williams, C.A. Smith, Molecular regulation of apoptosis:genetic controls on cell death, Cell 74 (1993) 777–779.

[98] H. Steller, Mechanisms and genes of cellular suicide, Science267 (1995) 1445–1448.

[99] A.M. Chinnaiyan, K. O’Rourke, B.R. Lane, V.M. Dixit, Inter-action of CED-4 with CED-3 and CED-9: A molecular frame-work for cell death, Science 275 (1977) 1122–1126.

[100] M. Oren, The involvement of oncogenes and tumor suppressorgenes in the control of apoptosis, Cancer Metastasis Rev. 11(1992) 141–148.

[101] U. lgbavboa, C.N. Zwizinski, D.R. Pfeiffer, Release of mito-chondrial matrix proteins through a Ca2+ requiring, cy-closporin-sensitive pathway, Biochem. Biophys. Res. Commun.161 (1989) 619–625.


[102] T.E. Gunter, D.R. Pfeiffer, Mechanisms by which initochondriatransport calcium, Am. J. Physiol. 258 (1990) C755–C786.

[103] C. Richter, V. Gogvadze, R. Laffranchi, R. Schlapbach, M.Schweizer, M. Suter, R. Walter, M. Yaffee, Oxidants in mito-chondria: from physiology to disease, Biochim. Biophys. Acta1127 (1995) 67–74.

[104] M. Leist, B. Single, A.F. Castoldi, S. Kuhnle, P. Nicotera,Intracellular adenosine triphosphate (ATP) concentration: aswitch in the decision between apoptosis and necrosis, J. Exp.Med. 185 (8) (1997) 1481–1486.

[105] C. Richter, M. Schweizer, A. Cossarizza, C. Franceschi, Con-trol of apoptosis by the cellular ATP level, FEBS Lett. 378(1996) 107–110.

[106] M. Oren, Relationship of p53 to the control of apoptotic celldeath, Semin. Cancer Biol. 5 (1994) 221–227.

[107] L.M. Adams, S. Cory, The Bcl-2 protein family: arbiters of cellsurvival, Science 281 (1998) 1322–1325.

[108] B. Antonsson, E. Conti, A. Ciavatta, S. Montessuit, et al.,Inhibition of Bax channel forming activity by Bcl-2, Science 277(1997) 370–372.

[109] E. Yang, S.J. Korsmeyer, Molecular thanatopsis: a discourseon the Bcl-2 family and cell death, Blood 88 (1996) 386–401.

[110] R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. Newmeyer,The release of cytochrome c from mitochondria: a primary sitefor Bcl-2 regulation of apoptosis, Science 275 (1997) 1132–1136.

[111] L. Soabo, M. Zoratti, The mitochondrial megachannel is thepermeability transition pore, J. Bioenerg. Biomembr. 24 (1992)111–117.

[112] L. Al-Nasser, M. Crompton, The reversible Ca2+ inducedpermeabilization of rat liver mitochondria, Biochem. J. 239(1986) 19–29.

[113] V. Petronilli, C. Cola, P. Bernardi, Modulation of the mito-chondrial cyclosporin A—sensitive permeability transition

pore. II. The minimal requirements for pore induction under-score a key role for transmembrane electrical potential, matrixpH, and matrix Ca2+, J. Biol. Chem. 268 (1993) 1011–1016.

[114] P.L. Gutierrez, N.R. Bachur, Free radicals in quinone contain-ing antitumor agents. The nature of the diaziquone (3,6-di-aziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) freeradical, Biochim. Biophys. Acta 758 (1983) 37–41.

[115] S.K. Jain, S.B. Shohet, Calcium potentiates the peroxidation oferythrocyte membrane lipids, Biochim. Biophys. Acta 642(1981) 46–54.

[116] D. Thomas, M.R. Hanley, Pharmacological tools for per-turbing intracellular calcium storage, Methods Cell Biol. 40(1994) 65–89.

[117] T. Kuwana, J.J. Smith, M. Muzio, V. Dixit, D.D. Newmeyer,S. Kornbluth, Apoptosis induction by caspase-8 is amplifiedthrough the mitochondrial release of cytochrome C, J. Biol.Chem. 26 (1998) 16589–16594.

[118] C.S. Scaffidi, S. Fulda, A. Srinivasan, C. Friesen, F. Li, K.J.Tomaselli, K.M. Debatin, P.H. Krammer, M.E. Peter, TwoCD95 APOA/Fas signaling pathways, EMBO J. 17 (1998)1675–1687.

[119] R. Hakem, A. Hakem, G.S. Duncan, J.T. Henderson, M. Woo,M.S. Soengas, A. Elia, J.L. de la Pompa, D. Kagi, W. Khoo, J.Potter, R. Yoshida, S.A. Kaufman, S.W. Lowe, J.M. Pen-ninger, T.W. Mak, Differential requirement for caspase 9 inapoptotic pathways in vivo, Cell 94 (1998) 339–352.

[120] K. Kuida, T.F. Hayder, C.Y. Ktian, Y. Gu, C. Taya, H.Karasuyama, M.S. Su, P. Rakic, R.A. Flavell, Reduced apop-tosis and cytochrome c-mediated caspase activation in micelacking caspase 9, Cell 94 (1998) 325–337.

[121] F. Cecconi, G. Alvarez Bolado, B.I. Meyer, K.A. Roth, P.Gruss, Apaf 1 (CED-4 homolog) regulates programmed celldeath in mammalian development, Cell 94 (1998) 727–737.

.

Oxidative Stress and Apoptosis

Documents

Transcript of Oxidative Stress and Apoptosis