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The importance of oxidative stress in apoptosis

Sharon Glutton

MR C Ra diation and Genom e Stability Unit Harw ell UK

In vitro studies have shown that apoptosis can be triggere d by many distinct and

different stimuli including exposure to physical and chemical agents or by the

removal of growth factors. Free radicals and oxidative stress have been implicated

in apoptosis, although there is much uncertainty regarding their importance.This

review aims to address much of the existing ambiguity.

Correspondence to.

Sharon Clutton

MRC Radiation and

Genome Stability Unit

Harwell Didcot

Oxfordshire OX ORD

UK

Apoptosis is a morphologically distinct form of cell death which plays akey role in embryogenesis and tissue homeostasis. The co-ordinatedultrastructural changes that typify apoptosis were first described by Kerretal

x. These are the compaction and marginalisation of chromatin in the

nucleus, accompanied by plasma mem brane blebbing and cell shrinkage,ultimately forming apoptotic bodies which in vivo are rapidly removedby phagocytosis by neighbouring parenchymal cells or by professionalphagocytes. Since the subcellular organelles remain intact and there islittle leakage of the contents of the dying cell, apoptosis provides theorganism with a safe method of maintaining genome integrity byallowing the organism to remove damaged or abnormal cells withoutcompromising neighbouring cells. This contrasts with necrotic cell death

caused by severe cell injury where the cells lyse releasing destructiveenzymes and potentially toxic chemicals, which may often causeinflammation.

There is increasing evidence that the molecular mechanism ofapoptosis has been evolutionary conserved since it occurs in bothnematodes and vertebrates. Of particular importance are the geneticstudies on the nematode Caenorhabditis elegans which have led to theidentification of two genes, ced-3 and ced-4 essential in developmentalcell dea th2. Mutation in either gene permits cells which would normallydie to survive. The ced-3 gene product encodes a protein which hasconsiderable homology with a family of mammalian cysteine proteases

related to interleukin ip converting enzyme (ICE)3-4. It is becomingincreasingly apparent that cysteine proteases play a key role in thedownstream events governing apoptosis in both nematodes andmammals. Resembling the observations in C. elegans overexpressionof a variety of cysteine proteases in mammalian cells has been found totrigger apoptosis5'6, while inhibition of their activity can protect thecells7'8.

©T he Brihth Councill997 Brituh Me dical ulletin 1997;33 No 3) 662-6 68



Oxida tive stresi in apoptosis

Oxidative stress

A diverse number of stimuli have been shown to induce apoptosis,many of them are also know n to compromise the fine balance betweenintracellular oxidants and their defence systems. Under aerobic

situations, the participation of oxygen in redox reactions is unavoid-able and a variety of highly reactive chemical entities are produced.These are comm only referred to as reactive oxygen species ROS) and

the list comprises the hydroxyl radical, hydrogen peroxide, lipidperoxides, nitric oxide and superoxide, to name but a few. Many of

these agents have a beneficial role in the cell but, when present in

excess, the cell becomes oxidatively stressed9. A number of grossbiochemical changes occur as a consequence of oxidative stress, the

extent depending on the severity of the insult. Extreme, non-

physiological concentrations of oxidants or oxidant generating agentscause necrosis rather than apoptosis, consistent with the hypothesisand observations of Duvall and Wyllie10 . Oxidative overload causesgross cellular damage resulting in alteration of redox state e.g.

depletion of nucleotide coenzymes, disturbance of sulphur containingenzymes), saturation and destruction of the defence and repairsystems. If the cellular balance is not restored, a number of

pathological processes are elicited. Predominant processes resultingfrom oxidative stress include oxidative lipid degradation (lipidperoxidation), the loss of intracellular calcium homeostasis and

alteration of metabolic pathways11 . All of these processes have beenrecorded in exhaustive lists of apoptotic models. However, an

interesting challenge remains to dissect out the biochemical effectorfrom the biochemical changes occurring as a consequence of celldeath.

The breakdown of membrane lipids increases the concentrationof biologically active lipids, such as arachidonic acid metabolites,and the release of diacylglycerols and ceramide from sphingomye-lin. These in turn can stimulate the influx of calcium and its releasefrom intracellular stores resulting in loss of calcium homeostasis on

which many integral enzyme systems depend. For example ,activation of calcium dependent poly-(ADP-ribose)-synthetase con-

sumes NAD, which con sequen tly l imits the availabil i ty of

NAD(P)H to take part in redox reactions, such as ATP and

glutathione generation12 '13 . Additionally, calcium-dependent endo-nuclease activation is believed to initiate the chrom atin fragmenta-tion frequently observed in apoptotic cells

14-

15. In association with

diacylglycerol release, calcium can activate protein kinase-Cdependent signal transduction pathways, thus modifying genetranscription 16 .

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Apoptosis

Involvem ent of ROS in apoptosis

Apoptosis first occurs during embryogenesis in the inner cell mass duringblastulation and, in a series of elegant experiments from Parchment andPierce17, hydrogen peroxide generated from amine oxidase activity was

identified in the blastocoel fluid as being responsible for this cell death.Their results were surprising since hydrogen peroxide was traditionallyassociated with pathogenic events including necrotic cell death.Similarly, toxicological studies revealed that exposure of cells to lowdoses of hydrogen peroxide triggered apoptosis instead of necrosis seento occur at higher doses18. Subsequently, a wide range of oxidants havebeen found to be apoptotic, these include organic and lipid peroxides,thiol oxidising compounds and redox cycling quinones

19-

20*

23'24

. More-over, oxidative changes have been found in a variety of cell types evenwhen non-oxidative stimuli have been employed, for example thymo-cytes treated with glucocorticoids or etoposide25 .

There is great confusion in the literature regarding the use of anti-oxidants as modifiers of an apoptotic response. In some systems there islittle doubt that free radicals and R OS are produced, such as exposure toionising rad iation, and so it is of no surprise tha t, in such exam ples, anti-oxidants have been shown to reduce or delay apoptosis. A good exampleof this is radiation and hydrogen peroxide induced apoptosis inthymocytes which can be inhibited by trolox, an inhibitor of lipidperoxidation25 , or reduction of apoptosis in blastocysts when treatedwith glutathione, N-acetyl cysteine or catalase17 . In other systems theoxidative insult is implied, such as after treatment with tumour necrosis

factor (TNF) or inflammatory cytokines (e.g. IL-1) which are believed toinitiate signal transduction pathways via ROS 26 . Again, agents such assuperoxide dismutase have been found to attenuate the effects

27.

However, the situation is ambiguous since the Fas antigen which isstructurally related to the TNF receptor can induce apoptosis whenincubated with the anti-Fas/APO-1 but does not readily respond toantioxidant treatment, although it may be that there is a divergentsignalling pathway between Fas/APO-1 and TNF receptor

28. Another

confounding problem with the use of exogenous antioxidan ts is that theymay also act as pro-oxidants depending on both the buffering capacity ofthe cell and the concentrations employed.

Is BCL-2 an an tioxid an t?

Perhaps the best studied negative regulator of apoptosis is the proto-oncogene product BCL-2. BCL-2 is a 26kDa integral membrane proteinoriginally cloned from the t(14:18) translocation breakpoint found in B-cell

66 4 Bnhih M d cal Bulletin 199723 No 3)



Ox ida tive stress in apoptosis

lymphomas. Expression of this protein has been found to rescue lethallyinsulted cells from a wide range of stimuli29 '30. BCL-2 is only one member ofa larger family of related proteins, some of which also prevent cell death,whilst others, like Bax, accelerate it. These related proteins can hom o- andheterodimerise, and the final commitment to death depends upon thestoichiometry of the effector and blocking proteins. The importance of thebcl 2 gene in apoptosis can be highlighted by the presence of a structurallyand functionally, related homologue ced-9 that plays a pivotal role inpreventing wide spread cell death in C. elegans

2.

Since free radicals have frequently been implicated in apoptosis,Hockenbery et at

23 tested the hypothesis that BCL-2 acted as an

antioxidant. Indeed they reported that sustained hpid peroxidation wasa feature of apoptosis induced by the redox cycling quinone, menedioneand by hydrogen peroxide, and both apoptosis and lipid peroxidationcould be modified by conventional antioxidants and also by over-

expression of bcl-2. Similarly, Kane et al3i

found that bcl-2 expressioninhibited apoptotic and necrotic cell death in neural GTl-7 cells treatedwith thiol depleting agents, and suggested that the bcl-2 gene productmodulated intracellular levels of free radicals, which had a knock-oneffect in suppressing apo ptosis. H owever, BCL-2 facilitated cell survivaleven after application of the thiol blocking agent diethyl maleate (DEM)and, therefore, it is unlikely that it could function as a direct free radicalscavenger. Perhaps the strongest argument against BCL-2 having directantioxidant properties came from experiments of Jacobson and Raff

32.

They demonstrated that prograrnmed cell death could occur when cellswere cultured at very low concentrations of oxygen, although not byagents known to exert their effects via ROS such as hydrogen peroxideand menedione. Similar experiments by Shimizu et at

33 failed to detect

ROS during hypoxia induced apoptosis. In both of these experiments,BCL-2 still offered protection against apoptosis. The mechanism ofBCL-2 remains unknown, although from these experiments it is clearthat BCL-2 is unlikely to act in the capacity as a free radical scavenger.Moreover, such evidence suggests that ROS are not the exclusiveeffectors in the death program me. The quest remains to find a com monpathway downstream of the possible initiating agents which unifies allobservations, and the relevance of oxygen independent redox reactions is

in question.

Intracellular redox state

The tripeptide glutathione plays an integral role in cellular redoxbiochemistry and, for this reason, the concentrations between reduced

Bnhth Medrnjl u»»hn 1997;33 No 3) 6 6 5



Apoptosis

Summary

References

and oxidised glutathione are stringently controlled. The machinery ofthe cell, the proteins, depend upon both the pH and the redox state oftheir micro-environment for their reactivity. For example, it is knownthat serine protease zymogens have a local requirement for high localconcentrations of oxidised glutathione34 . In addition, protein-thiol

modification is believed to be involved in the initiating steps for proteinproteolytic cleavage

35. Activation of the ICE-like enzymes which are

believed to orchestrate apoptosis, requires proteolytic processing, whichhas lead to speculation that oxidative thiol modification of ICE-likeenzymes may be integral to controlling apoptosis

36. As yet, there is only

indirect evidence to support his hypothesis.Agents which disrupt thiol homeostasis, such as DEM and (d-1)-

buthionine sulphoximine have been shown to induce apoptosis, whichcan then be rescued by agents that restore this balance 21 23-33.Intracellular glutathione levels also drop during apoptosis when both

oxidative and non-oxidative stimuli are applied, and only in the case ofapoptosis perpetrated by ROS has there been a conco mitant increase inoxidised glutathione37 . Intriguingly, Slater et al

36 found that protease

inhibitors can preven t both apoptosis and g lutathione efflux. In search ofa common pathway for apoptosis, investigators have suggested thatBCL-2 may play a central role in intracellular signalling by regulatingcalcium38 or glutathione efflux37.

There is no doubt that oxidative stress can elicit cell death, and that mildoxidative stress can initiate apoptosis rather than necrosis; althoughreactive oxygen species can cause oxidative stress, they are not essentialfor the apoptotic processes to occur. ICE-family proteins play the part ofcellular executioner, but the biochemical messengers remain to bedetermined. A tempting, unifying hypothesis is that perturbation ofcellular redox homeostasis may control these key events.

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