Dna Damage By Ros

R?r?cciveti

I I Beccnrbcr1990;rcviacd vcrrian rcccivcd 22 J~nurrry1991

When erllw arc rxpurcd to uxidativc xwcxx. DNA ddmapc rreyucnrly ~lccurx. The mulcrul~r mcchonixmr euurhu this damrgr mry include acrivritien or nuclsnxcx crnd direcr rcw.4on af’hydrasyl rndicelx with thr DNA. Scvcrw~ axygcn=drrived rpcciex ten rt~nck DNA, pruclucing dixtinclive pattetna aPshcmiet~l madificcrrian. Obxwution of ~herc pwternw and mwarcmcm ul’ xamc ui’ the products i%rmcd hax &en uwrl (a dctcrminc the rele nT difkrcnt axygetwierivcd rpccicx in DNA elr:~vagc rcnctionx, w LIIJCS rhc extcnf uf oxidalivc dnmrgc to DNA in viva and tc) invcsliptitc lhcr mcch:mixm of DNA dww\gc by iuoixing redi&n nnd rhcnrieal careinugcnx.

I. INTRODUCTION It is well-esrablished rhar aerobic organisms conxrantly produce small amounrs of reactive oxygen species’, including supcroxidc radical (OF), hydrogen peroxide (HZ@) and hypochlorous acid (HOCl), the latter being generated by the enzyme mycloperoxidase in ncutrophils [l-3]. Exposure of living organisms to background levels of ionizing radiation leads to homolytic fission of oxygen-hydrogen bonds in water to produce hydroxyl radical, ‘OH [4], Hydroxyl radical can also be generated when Hz02 comes into contact with certain transition metal ion chelates, especially those of iron and copper [S], In general, the reduced forms ,of these metal ions (Fe2’“,Cu”) produce ‘OH at a faster rate upon reaction with Hz02 than the oxidized forms (Fe’+) Cu2’) and so reducing agents such as 01 and ascorbic acid can often accelerate ‘OH generation by metal ion/H2Oz mixtures [S]. However, both Cu2* [6,7j and certain Fe3”-complexes (especially Fe3*-nitrilotriacetic acid) do generate some ‘OH upon raction with Hz02 [$-IO], In the case of ferric-NTA address: 13. Halliwell, Division of Pulmonary Medicine, UC Davis Medical Center, 4301 X Street, Sacramento, CA 95817, USA Correspottdmce

’ ‘Reucrive o_~_~gen species’ is 2 collective term used in the biomedical literature that includes oxygencontaining radicals (such as Of, ‘OK, RO’, R02’) and non-radical species that can produce oxygcncontaining radicals during their reactions (HzOz, 03, singlet OrlAg, HOCI). The term ‘reactive’ is relative; for example, Oi is very much less chemically reactive than is ‘OH.

Published

by Ehevier Sciettce PublMers

B. V.

complexes, Or is somehow involved in the ‘OH Formation in rhc presence of HZ&, since it is almost completely inhibited by the O:-scavenging enzyme superoxidc dismucase [9,10]. Aerobes have evolved antioxidant defences to protect thcmsclvcs against the reactive oxygen species’ generated in viva, These clefences include enzymes (such as supcroxide dismutasc, catalast: and glutathionr pcroxidasc), low molecular mass agents (examples being cr-tocopherol and ascorbic acid) and proteins that bind metal ions in forms unable to accelerate free radical reactions [ 1,2,1 l-l 31. Oxidative sfress results when reactive oxygen species are not adequately removed. This can happen if antioxidants are depleted and/or if the formation of reactive oxygen species is increased beyond the ability of the defences to cope with them [2]. Subjecting cells to oxidative stress can result in severe metabolic dysfunctions, including peroxidation of membrane lipids, depletion of nicotinamide nucleotides, rises in intracellular free Ca** ions, cytoskeletal disruption and DNA damage, The latter is often measured as formation of single-strand breaks; double strand breaks or chromosomal aberrations. Methods for measuring DNA strand breaks have recently been discussed [14-161. Indeed, DNA damage has been almost invariably observed in a wide range of mammalian cell types exposed to oxidative stress [16-441. The systems used to impose oxidative stress upon cells have included exposure to elevated oxygen concentrations [42], incubation with enzymes that generate reactive oxygen species (such as xanthine oxidase plus its substrates, xanthine or hypoxanthine [18,22-27]), 9

rllircct ;additien to the exllxp al H$~z [i8,2&=32,43,44, 44e,bf, sPsrgsrnichydr~peroxidem [3%36,44~1lr,45], or of ceinpound~ whaae metakmlism by the?cell ~eswltr in in= creased, inrraeeliuirr g~r~~r~ti~n ST Oi and H$& (such. msparaquat tend mcnndieane (LOJI:]}, and c&ncubation of the eclls with de-tivrtcd phagacytes such PJ mncrephr#cs [38) and neutrophiis [18,39-411, Ar. civnted ncutrophils and tntlcrophages generate 0: and HrOp: in addition, +utrephiis produce HOC1 [Zjr46), Neutrophils do not produce *OH unleaa a s~urcc OF rransition metal ions is added to the incubation mlxcure, ix. neutraphils do not them&es appear to can. tain nny form of metnl ion cntalyse that will convert HaOr into ‘OH [47-49)‘. Oxidative stress [XJ-541 and DNA dnmagc [51J also occur when some mammzGtln cclis are exposed to tumor necrosis factor. The DNA damngt: produced in human cetis by exposure to cigarette smoke [55,55a,56], asbestos [57,58), ozone (60,61 J or to ecrtain carcinogenic met&, such as nickel (591, haa also been suggested to involve reactive oxygen specks, OF DNA DAMAGE INDUCED BY OXIDATIVE STRESS

2. POSSIBLE MECHANISMS

Why does oxidative st ress cause DNA damage? In the case of extcrnaiiy-generated reaccivc oxygen species (e.g. when cells arc incubated with HzOZ, activated phagocytes or xanthine oxidase plus its substrates) damage is usually inhibited, by adding cataiase, showing that Hz02 is needed. Superoxide dismutase (SOD) does not usually inhibit much, which could mean either that Or’ is not involved in the DNA damage, or that SOD does not enter cells easily. That the tatter interpretation is correct in at least one cell system is shown by the observations that SOD can protect hepatocytes from the toxicity of Hz02 or f-butylhydroperoxide under conditions where it does enter the cells [62,63], However, neither 0; nor Hz02 undergoes any chemical reaction with DNA, as measured by strand breakage [64-661 or by chemical changes in the deoxyribose, purines or pyrimidines [9,67,68]. Hence, DNA damage by oxidative stress cannot involve direct attack of 01 or of H202 upon the DNA. Two explanations of the DNA damage have been advanced (Fig. 1). First, it is possible that the damage is due to ‘OH radical formation [30]. Thus, it is envisaged that M202, which crosses biological membranes easily [69J, can penetrate to the nucleus and react with ions of iron or copper to form ‘OH. Because of the high reactivity of ‘OH and its resultant inability to diffuse significant distances within the ceil [69], this mechanism is only feasible if the, ‘OH is generated from Hz01 by reaction with metal ions bound upon or very close to the DNA. One possibility is that these metal ions might always be present bound to the DNA in vivo. For exampie, copper ions are thought to be present in 10

~hr~m~%~rn~~I781 tend copper iona &re very cffeetivo in promoting H~~~-d~p~n~l~nt dramage to iaolnted DNA t&7) ana 5~ DNA within chramtstin [71J in vitrs, A x1?erand ~~$~ibility ir thert ths mcttttl iana might be releasrd within the crll w U nrutt of oxidntive mess, mt then bind to the DNA /71]. Thus, just ns oxidntive stress EBWILC?L rises in intr~ellula~ free,Clrrc, it rntxy cause rises in intraccllulrr Prcc Iron and&r copper ions that could bind to DNA and make it w target far oxidative dtrmnp

f72-741, It hna recently been shown chat game chelators 00 the carcinogenic metal nickel [59] also react with HZ@&to cause ‘OH-dependent damage to isolated DNA, Mixtures of eobnlt(IIj ions and &Or which arc thought to produce ‘OH [f!], again damaged DNA in a way characicristic of attack by ‘OH (Nzlekerdien, Rata, Haliiwcll, and Dizdaroglu, in preparation, A second cxphnation of the ability of’oxidativc stress to CQIISCBNA damage is that it triggers off a series of

metabolic events within the cell 176,771that lead to activation of nuclcasc enzymes, which cleave the DNA backbone. There has been muc.h debate recently concerning the suggestion that oxidative stress causes rises in intracellular free Ca’“, which might fragment DNA by activating Ca”*-dependent endonucicases [21,37,78] in a mechanism resembling that of apoptosis (‘programmed cell cieath’), An example of apoptosis is the killing of immature thymocytes by glucocorticoid hormones, which activate a self-destructive process that apparently involves Ca’*-dependent DNA fragmentation [79,80]. These two mechanisms (DNA damage by ‘OH or by activation of nucleases) arc not mutually exclusive, i.e. they could both take place. Indeed, there is evidence consistent with both of them. Their relative importance may depend on the cell type used and on how the oxidative stress is imposed. For example, chelating agents that bind iron ions into chelates unable to generate “OH (such as desferrioxaminc [SlJ, desferrithiocin 1181,or phenanthroline [82]) can often protect ceils against DNA damage and other toxic effects of oxidative stress [30,31,34,38,73,83-$51, The effects of desfcrrioxamine are variable, since in general it does not cross cell membran.es readily, although it appears to enter some cell types (such as hepatocytes) more readily than it enters others. Jonas et al. [29] showed that the toxicity of I-3202to epithelial cells is greatly diminished at 4°C but it can be increased again by adding ascorbic acid: this effect is not seen if cells are pre-treated with desferrioxamine. Their observations are consistent with a mechanism of cell damage that depends on Hz02 and reduced iron ions: at high temperatures normal metabolism may provide a reductant (such as OF), tihereas at low temperatures ascorbate can replace it. Suggestive evidence that Fenton-type reactions can occur within bacterial ceils has been presented [87-$93, although this is not necessarily relevant to mammalian systems. SHydroxyguanine (&OH-Oua) was increased in amount in’the DNA of P388 Dl cells after exposure

Fig. 1, Myporlrcrcs to cxplnin DNA

dnmngc resulting from exposing cells to oxihiw

to I-I202 [18]: d-OH-Gua can arise from attack of ‘OH upon guanine (see next section) but should not be produced as a result of nuclease action. DNA isolated from P388 Dl cells after exposing them to Hz02 did not show a regular pattern of fragmentation, such as might be expected from nuclease attack [ 181. Increases in g-OHGua have also been observed in DNA from other cells subjected to oxidative stress [90,91]. Thymine glycol, another product that can result ,from attack of ‘OH upon DNA (see next section) has been reported to be formed in the DNA of yeast cells after exposure to high concentrations of I-I202 [92] and in murine tumor cells after exposure to tumor necrosis factor [S11. Treatment of murine hybridoma cells with Hz02 caused a pattern of chemical changes in the DNA bases that is characteristic of attack by ‘OH [93].

StrCs.s.

However, the evidence for metabolic changes produced in cells by oxidative stress is also strong [21,39,96-991. Menadione and other quinones (which ‘redox cycle’ within cells to give 02 and H202) appear to produce DNA strand breaks in hepatocytes by Ca2+-dependent activation of an endonuclease. DNA damage could be inhibited by preventing the rise in Ca2+ using Ca 2+-chelators [37,94]. Oxidative stress can also sometimes activate and/or cause changes in the subcellular location of PKC (protein kinase C) [95-993. Exposure of mouse epidermal JB6 cells to Hz02 appeared to cause a Cazc-dependent translocation of PKC to the piasma membrane [!X] whereas menadione activated PKC both in these cells and in rat hepatocytes [95,963 without producing translocation. Cantoni et al. [98] found that the Ca2’ -chelator quin 2 inhibited

Can mutationa lnduecd k314* t3xSdnrlvt: mw lasd ttl can&! IcMxing radiscion k well-known to bc both mutagenic and ~ar~~l~o~~nj~ (&I 10, I t I]. Since much crf

the CC\! darnqc eauasdby xuchradiatian invslvcr ‘OH preduetien by kemelytic fisrian al rhcr tlxygen= Fig, 2 trhow,a that both type% of experimental result may be nccommodnred by proposing that ohungca in the availability of calcium lona may depend upon, or give risa 10, changer in the availnbility oi+ iron or copper ions, Clearly, attempting co elucidate t;tvz mfchanism af DNA damage by thr use of free radical scavengers or metal ion chelators added to the outlsidc of ucllt ix

unlikely 10 give unambiguous answers. Let 11ssee what can be learned from the techniques of molecular biology and analytical chemistry. 3. REACTIVE OXYGEN AND CARCINQGENS

SPECtES A$ MUTAGENS

Oxidative stress, imposed by a variety of mechanisms (including increased 02 concentrations [98a]), has been convincingly shown to be mutagenic to bacteria [69,99-1031. For example, E. coli mutants bxking SOD activity show greatly-enhanced rates OF spontaneous mutation [99]. Similar mutagenic effects have been shown in a range of mammalian cell types (42,105-1071 subjected to oxidative stress. Moraes et al. [108] studied the pattern of mutations obtained in a gene of a shuttle plasmid when simian cells transfectcd with this plasmid were exposed to HzOZ. Both single base changes and deletions were observed. The majority of base changes were at GC base pairs, the GC-+AT base transition be-

Fig. 2. A combined

12

hypothesis. Rises in intracellular

hydroycn btlnda in waFerI then ‘GH can grabrbly bi: clnssifitld as a eompizrs carcinogen, Bascqair changes and Some frt?rnr&Ma are the eemrnoncot murririsns observed in cells exposed to ionizing radiation (I 10, it I], Chemical ckangcs In the: 5NA bases, aingicand double-strand breaks and cnhnnecd cxprcs&on of certain prote-oncogcner [&I 12,113] have also been observed. WOwever, the prceise relationship between these differenr events tend rhc dcv~lopmttnt of cancer is uncertain. ‘i’hur, the chemical changes in DNA might themselves somehow lead to cancer [ LILE]. An unrepaired lesion in DNA mighr lx by-pasaccl in an error-prone fashion. Resynthesis of BNA after excision repair might conceivably introduce errors. Thcrt: are many steps between a healthy cell and a malignant tumor, Cancer biologists have often referred to at least three stages: initiation (an irreversible change in DNA), pramotion (probably involving changes in gene cxprcssion) and progression (further changes in DNA leading to the cvenrual production of a malignant tumor). Both Zimmerman and Ccructi [115] and Weitzman et al. (1161 showed that a clone of C3H mouse fibroblasts exposed to activated human ncutrophils or to hypoxanthine plus xanthine oxidasc underwent malignant transformation* Nassi-Calo et al. 1117) showed that Hz02 also transformed these cells, an action prevented by the chelating agent o-phenanthrolinr.

free iron or copper ion concentrations vice versa (IA, 28).

could be a consequence of rises

in Ca”*

(lB, 2B) 01

Ahhougk

mw

r\trcnricw

haa ken

paid in ths mypen rpceicr yIx pro-

lircraturr! to I hi(Eacticsn of rewtivf mw33 of cawcinagencsia [ 115,120,121,123], their ablli-

ty fo dam~tgc DNA and produce alrerarionu~in gcnc ex= prexxion implies that they could bc involved in all srager af careinogeneais [124,127,129]. It bar been argued [42,130- 132] that canrinuoua: dama~? to DNA by free radical mechanisms ia B significant ERWJCof cftnccr in humans, nn abwvation rlw might explain rcaultn of the epicicmiologiral investigations, which show invlersc corrclarions in humans between plasma concentrations

of certain an~ioxidants and rhe: incidence of cancer [ 131,133], However, several anrioxidnnfs can alter the metabolism of procarcinagcns, Favoring metabolic pathways rhar do nat result in formation of ultimate carcinogens [ 13I ,134]. Menee a protective effect OFantioxidants does 1101r~ecesstrril,~mean rhnt oxidtlrivc Stress leads fo the cancers in question. Of course, some carcinogens might act by imposing s?n oxidnrive stress on their target cells (Section 4), and reactive oxygen species have been claimed to be capable of converting some procarcinogcns into ultimate carcinogens [134,135]. In addition, it must be borne in mind thar a high plasma level of such antioxidants as ascorbic acid and vitamin E may simply be an index of a good diet, which protects against many diseases. DNA damage resulting from oxidative stress (or from any other mechanism) need not necessarily lead to cancer. Low lcvcls of damage may be efficiently repaired with a minimal risk of error, High levels of oxidative stress may lead to cell death, so that initiated cells do not remain in the organism. Thus, an intermediate level of damage is most likely to predispose to malignancy, which may explain the close association of chronic inflammation (involving phagocytic production of 02 and H202) with malignancy in such human diseases as ulcerative colitis, Crohn’s disease and reflux esophagitis (reviewed in [127]). Cerutti et al. [122] showed that one difference between a clone of mouse epiderrnal cells that was promotable by xanthine/xanthine oxidase and a non-promotable clone was that the latter had lower levels of SOD and catalase and was more sensitive to killing by reactive oxygen species. Thus, increased antioxidant deferices, by protecting against cell death resulting from oxidative stress, might conceivably (and ironically) sometimes lead to increased cancer [136,137].

Radiation chemists have carried we many rrudicP of the effcct~ of ‘OH, yencrnred by ionisingg radintion, upon DNA, Thix radictt! ia rotarrsctive that it can attack ail csmgoncntx of the DNA (reviewed in (4,138-1403). Thus, ‘OH abstracts hydrogen atoms Cam dwxyribosc, giving su&ar radicals that can fragment in various ways. Reactions of deoxyribose-derived radicals can lead to lhe release of’ pwine nnd pyrimidine bnses from the DNA (producing abasic sites), and to strand breaks. Some of the altered sugars that rrmnin artnchcd to DNA can be xplir to give strand breaks by

incubation with alkaline solutions; these are the socalled “alkali-labile sites [4,15,141]. Chemical changes to rhc purine and pyrimidinc bases have also been characrerized in detail (reviewed in [ 138-140)). Thus, ‘OH can adcl on to guanine residues at C-4, C-5, and C-8 positions. For cxamplc, addition OF ‘OH to C-8 OF yuaninc produces a radical adduet rhat has scvcral possible fates. Ir can be reduced to Ghydroxy-7,8-

dihydroguanine, osidized to &hydroxyyuaninc, or undergo ring opening followed by one-electron reduction and protonation to give 2,G-diamino-4-hydroxy-Sformnmidopyrimidinc, usually abbreviated as FapyGua (Fig8 3), Similarly, ‘OH can add on to C4, CS, or C8 of adcninc residues: among other fates, the C-8 ‘OH adduct radical can be converted into 8-hydroxyadenine by oxidation, or undergo ring-opening followed by oneelectron reduction to give 5-formarnido-4,6-diaminopyrimidine (FapyAde). Fig. 4 shows the structures of sonic of these compounds. Pyrimidines in DNA are also attacked to give multiple products. Thus, thymine can form c/s and tram thymine glycols (5,6-dihydroxy-6hydrothymines), %hydroxy+methylhydantoin, 5,G-dihydrothymine and 5-hydroxymethyluracil. Cytosine can form several products, including cytosine glycol and 5,Gdihydroxycytosine (Fig. 4). When whole cells or isolated chrotnatin are exposed to ionizing radiation, cross-links can occur between DNA bases and amino acid residues in nuclear proteins [139,142-1481. Thus, thymine-tyrosine [ 1451, thyminealiphatic amino acid [ 143- 1491, and cytosine-tyrosine [ 1463links have been identified in isolated calf-thymus chromatin subjected to y-irradiation. Treatment of chromatin with Fe”‘-chelates and Hz02 also produces DNA-protein cross-links [153] and such !inkc,have been detected in cellular DNA after exposure of the cells to ozone [60]. Molecular biologists have examined the likely 13

physiological effects of these various lesions in DNA, GHydrsxyguanine (and, by inference, &hydroxyadenine) might lead to mutations by inducing misreading of the base itself and of the adjacent bases [llO,ll 1,151,152]. Thymine glycol might have some mutagenic action and it can be lethal if not removed from the DNA before replication [ 110,ll l], Rinyfragmcnccd bases are thought co block DNA replication [I 10,ll I]. Abasic sites, which can result from direct attack of ‘OH, can also be mutagenic in vivo [I 10,l 111, It is clear chat ‘OH products multiple changes in DNA whereas 02” and Hz02 have no effect, but the

S-hydroxy-fi-mthyl-

situation with other reactive oxygen species is less clearcut at prcscnt, Sinylac oxygen is able I0 produce limited strand breakage in isolated DNA [153,154], and its ability co modify the DNA bases is aIso limited (154a]. Thus, M. DizdnrogIu and H. Sits (personal communication) found small amounts of d-OH-Gua and FapyGua but no ocher significant base changes in DNA exposed to singlet 0~ gcneratcd by the thermal dccomposition of an endopcroxide. Exposure co illuminated Mechylene blue causes formation of &hydroxyguaninc [155] and of some strand breaks [I561 in DNA but the species responsible was not identified, except for the

5Aydroxyurocil

S-hydroryhydmtofn

hydmtoin

S-hydtoxymotbyluracil

4,G-diamLm-5Pormmidopyr@ddina

S-hydroxycytoslne

8-hydroxyadcnino

Fig. 4, Some of the end-products

14

thymine

elycol

5,6-dihydcoxyuracil

2,6-dlamfno-4-hydroxy-

5,6-dihydroxycytosinc

8-hydroxygunnine

5-fomiamidopyrimidinc

that result from attack of hydroxyl

radicals upon the bases of’DNA.

Enbiefvationthar rcavenflarr at ‘OH did not protccc.

Hawcver,dha excited state of the ph~to~en~iti~~n~ ,dya Raw&2bengIl hWKiwlf been clrSmed ta clewve DNA [ 157)

and, if ‘OH were $anerurcd by Mcthyirne blue bound to the DNA, then ‘01-I srcavenfi~r% would nar b+ experfcd 10 pmteef. Elluminated ribcafhwin, which aenrratca

rinatet CL WWJ regarted no1 to pradusc I-hydrtlxygurninc in DNA [l%t]. Thus, sin@et 8: certainly does nat induce the extenrive pattern af DNA base modifieta* tion produced by ‘a-s. Peroxidixing lipids hwe been reported to dxlmnp DNA flS9=163a] but peroxidizing lipids produce a rengc of reactive oxygen species including ‘OH, W&II, singlet oxyycn, peroxyl radicnls, and nlkoxyl radicals [69,164] i0xl the exact contributions of these species to the DNA damage observed need to be determined [165,166]. Lipid peroxides alno decompose to give tl huge range of producta [166-1683 ineluding carbonyl compounds, such na maiondia~dehyde and the unsaturated tlldchyde 4-hydroxy*2-lrnrrs nonenal [ 168]

which has been shown to be mutagenic to mammalian

cells [ 1691. If these aldehydcs arc generated in the vicini-

ty of DNA, they mlry be able to combine with it to form distinctive products [ 165,169], Thus, malondialdchydc reacts with adcnine, eytosine, and guaninc [161,17011] and a guaninc-MDA adduet has been identified in human urine [ 1711,The product of reaction of hydroxynonenal with deoxyguanosinc has also been characcerized [172]. Humans arc constantly exposed to background levels of ionizing radiation, which will generate some ‘OH in vivo. This radical may also arise by reaction of metal ions with H;rOzin vivo [5]. Thus, it is not surprising to find that repair systems have evolved to remove at least sotne of the lesions in DNA that can result from attack of ‘OH and other reactive oxygen species (reviewed in [ 173,174]). Single-strand breaks, ate usually quickly repaired; indeed, they are generated as intermediates in the repair of other lesions (see below). 8-Hydtoxyguanine is slowly removed from cellular DNA, but the repair mechanism is unknown [ 1731.Several lesions, including thymine glycol, are probably removed in human cells by action of a DNA glycosylase, which cuts the base-deoxyribose bond to give an abasic site. This site is recognized by an endonuclease activity (on the same enzyme), which nicks the strand at the abasic site. The damaged part of the strand is removed, followed by resynthesis of the DNA and re-joining of the strand by a DNA ligase enzyme. Glycosylases that recognize hydroxymethyluracil and ring-opened purines in DNA have also been described in mammalian cells [110,111,173,1743. Modified DNA bases and nucleosides (base-deoxyribose) have been detected in the urine of humans and other mammals. Thus, S-hydroxyadenine, 7-methyl-8 -hydroxyguanine, thymine glycol, thymidine glycol, hydroxymethylutacil, 8-hydroxyguanine and B-hydao-

xyd~o~~~~~~n~~~n~ huve bet?n detected in rn~rnrn~t~~n urine [f35,175-L781. The prcwxe erf these produan In urine ~~~~~e~r~that axidativc damage to t hc fS‘NA bwec~ daes cxcuf In viva, and thwr repair nyatcnx ure wetiw to

cleave modified b~tscir from DNA, ~“~OVI”CVC~, it ia pnnsitale! that xomc excreted bases ori&ate from the dicr or from the mernbralism af rho gut flora, and chat DNA relewd fram dead and dying cells within an erganlsm undergo+% rcyitl oxidaticr drmaae (since cell disruption can incrensc Free radical reaetions [69,72,164]). Hence, one must be eaurious in using the amounts af modified

DNA brracaexcreted ftam the body tls an index of thr extent of repair of oxidative eclls.

DNA

damage in hartlrlr~

5. MEASUREMENT OF BASE-DERW33 PKXGJCTS AS A PROBE FOR THE

MECHANISM AND EXTENT OF DNA DAMAGE

The development

of an I-WK

tcchniquc,

coupled

with highly-sensitive clcetroehemical detection, for the measurement of &hydroxy-deoxyguanosine has led to a

series of pioneering studies in which measurement aF this product has been used to gain information about free eadical damage to DNA in intact cells and whole organisms [91,114,179-1811. Antibody techniques for the measurement of such products as &hydroxyguanine and thymine glycol have also long been available (e.g. see [182-184)). Ames et al. [175-1771 have used the urinary excretion of products derived from guaninc and thyminc as an index of free radical damage to DNA in vivo, and have attempted to draw conclusions about changes in the rate of such damage as a function of age in mammals. The amount of 8-hydroxyguanine in the DNA from certain sub-populations of eat liver mitochondria was found to be considerably higher than that in nuclear DNA, leading to proposals about the role of mitochondria in aging and carcinogenesis [ 185,186]. Exposure of some cells to oxidative stress has been reported to lead to formation of Wydroxyguanine in the DNA [22,90,91 ,114]. For example, treatment of Ehrlich ascites cells with the carcinogen 4-nitroquinoline l-oxide led to an increased content of 8-hydroxyguanine in DNA [187]. Intra-peritoneal injection of ferric-nitrilotriacetic acid (which reacts with HZOZto give ‘OH [9,10]) into rats produced a significant rise in the %hydroxyguanine content of kidney DNA [lSSJ. Carcinogenic peroxisome proliferators [189,190], acetoxime [191], 2-nitropropane [191] and, in one study, a choline-deficient diet [lgla], have all been reported to result in increased amounts of 8-hydroxyguanine in DNA in vivo in mammals. These studies have certainly produced qualitative evidence for oxidative damage to DNA in vivo, although care must be used in interpreting the data [191b]. For ex\mple, nitroquinolines have been sug-

Valum~ 261 nwmkr I

I ,S

FRFS

yeated m

form ~-hy~~rox~~~tanine in DNA by II mc?chanhn thut involves direct reacrion %rfthe utrimetc eareinsgcn with RNA, rarhcr than by oxygen radical gencrarion [192], Qne must also bs exrremely cautious in awzmpring to use measuremcnl of any one product ax a quttntifnriva measure of DNA base damage by rcacrive oxygen species. When ‘CM attacks DN4 bases, radicals are formed that can react in various ways depending on rhc candiriona used (Fig, 3 shows an cxample), Thus, acre& of ‘OH upon gunnine an lead IO fornwien of 8.hydroxyyunnine by oxidation of rhe C-8 ‘OH adduer radical, bur rhia radical can lead to orher products as well, depending on the reaction conditions. Thus, variable atnounts of &hydroxyguanine can resulr from attack of rhf same amount of ‘OH upon guaninc in DNA, and so changes in 84lydroxyfluanine levels do not necessarily mean changes in the amount of free radical attack upon DNA. To take some examples, iron-ion dependent systcri~ generating ‘OH led to substantial fortnarion of FnpyGua as well as 84tydroxyguaninc in DNA (671, whereas systems containing copper ions and Hz01 greatly favored 8-hydroxyguaninc production [6,7 I, 1931. When isolated, mammalian chromatin was irradiated in aqueous suspension, the relative atnounts of 8-hydroxypurines and formamidopyrimidines generated depended upon the radical environment provided by the gases used to saturate the aqueous solution [194]. For example, the presence of oxygen favoured the formation of %hydroxypurines [19$196]. Table I summarizes some of rhc results obtained, Products derived from pyrimidincs can similarly bc affected by changes in reaction conditions [195,196]. A complete characterization of damage to DNA by reactive oxygen species can be achicv,ed bg tl;; tcchnique of gas chromatography/mass spectrometry (reviewed in [194a,197,198]), which may be applied to DNA itself or to DNA-protein complexes such as chromatin. The DNA or chromatin are hydrolyzed and the products .converted to volatile derivatives, which are separated by gas chromatography and identified by mass spectromctry. High sensitivity of detection can be achieved by operating the mass spectrometer in the selected ion monitoring (SIM) mode. In this mode, the mass spectrometer is set to monitor several ions derived by fragmentation of a particular product during the time at which this product is expected to emerge from the GC column. The GUMS-SIM technique is being used in the authors’ laboratories to examine the mechanism by which DNA is damaged in cells subjected to oxidative stress. Thus, if damage is due to ‘OH generation, then products characteristic of ‘OH attack should be detected (Fig. I), as has been observed in murine hybridoma cells treated with Hz02 [93] and in primate trachea1 epithelial cells exposed to ozone (Aruoma, Halliwell and Wu, in preparation). By contrast, cleavage of the DNA backbone by the action of 16

0.55 I,8 0.8 3.4

0.57 3.5 0.95 4.5

nucleases should leave the purines and pyrimidines unaltered (Figs. 1 and 2). For studies on DNA modification, extraction of chromatin from cells for analysis is preferable to extraction of DNA, since it minimizes the loss of extensively-fragmented DNA, and of DNA that has becotne covalently cross-linked fo protein. GUMS-SIM has been used to characterize the damage done to DNA by various reactive oxygen species. Hydroxyl radical appears to produce a uniquely-extensive pattern of base modifications (multiple products from all four bases), cind this pattern seems to be a ‘fingerprint’ for ‘OH, i.e., it can be used to identify ‘OH as a damaging species [6,9,68, 71,139,143,146,152,193,194,196,198,201]. For example, measurement of damage to the DNA bases by GUMS-SIM has been used to show that the strand cleavage produced in isolated DNA by treatment with a copper-ion phenanthroline &elate probably involves ‘OH [ 1931,whereas DNA cleavage by a bleomycin-iron ion complex is not mediated by ‘OH [ 1991.GUMS has also been used to identify &OH&a, FApyAde, 8-OHAde and FApyGua in neoplastic tissues [200], to show that the damage done to the bases in isolated DNA by activated human neutrophils is most likely due to ‘OH generated by reactions involving metal ions in the reaction mixture [2Ol], to measure adducts of carcinogens with proteins in vivo in attempts to assess carcinogen exposure [202] and to characterize the changes produced in plasmid DNA by treating it with potassium permanganate [203]. The authors believe that such

Dypbukt, J,M.,

Thor,

H.

nnd

Nirtlldrn,

Rrrtlirttl Rcs, Carn~~~ur~,6, 34&&W, Chang, Y&Z., Hcppner, G.H., RIIII,

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