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 complexes, Or is somehow involved in the ‘OH Forma-

tion in rhc presence of HZ&, since it is almost complete-
It is well-esrablished rhar aerobic organisms conxrant- ly inhibited by the O:-scavenging enzyme superoxidc
ly produce small amounrs of reactive oxygen species’, dismucase [9,10].
including supcroxidc radical (OF), hydrogen peroxide Aerobes have evolved antioxidant defences to protect
(HZ@) and hypochlorous acid (HOCl), the latter being thcmsclvcs against the reactive oxygen species’
generated by the enzyme mycloperoxidase in generated in viva, These clefences include enzymes
ncutrophils [l-3]. Exposure of living organisms to (such as supcroxide dismutasc, catalast: and glutathionr
background levels of ionizing radiation leads to pcroxidasc), low molecular mass agents (examples be-
homolytic fission of oxygen-hydrogen bonds in water ing cr-tocopherol and ascorbic acid) and proteins that
to produce hydroxyl radical, ‘OH [4], Hydroxyl radical bind metal ions in forms unable to accelerate free
can also be generated when Hz02 comes into contact radical reactions [ 1,2,1 l-l 31. Oxidative sfress results
with certain transition metal ion chelates, especially when reactive oxygen species are not adequately remov-
those of iron and copper [S], In general, the reduced ed. This can happen if antioxidants are depleted and/or
forms ,of these metal ions (Fe2’“,Cu”) produce ‘OH at if the formation of reactive oxygen species is increased
a faster rate upon reaction with Hz02 than the oxidized beyond the ability of the defences to cope with them [2].
forms (Fe’+) Cu2’) and so reducing agents such as 01 Subjecting cells to oxidative stress can result in severe
and ascorbic acid can often accelerate ‘OH generation metabolic dysfunctions, including peroxidation of
by metal ion/H2Oz mixtures [S]. However, both Cu2* membrane lipids, depletion of nicotinamide nucleo-
[6,7j and certain Fe3”-complexes (especially tides, rises in intracellular free Ca** ions, cytoskeletal
Fe3*-nitrilotriacetic acid) do generate some ‘OH upon disruption and DNA damage, The latter is often
raction with Hz02 [$-IO], In the case of ferric-NTA measured as formation of single-strand breaks; double
strand breaks or chromosomal aberrations. Methods
for measuring DNA strand breaks have recently been
Correspottdmce address: 13. Halliwell, Division of Pulmonary
Medicine, UC Davis Medical Center, 4301 X Street, Sacramento, CA discussed [14-161. Indeed, DNA damage has been
95817, USA almost invariably observed in a wide range of mamma-
lian cell types exposed to oxidative stress [16-441. The
’ ‘Reucrive o_~_~genspecies’ is 2 collective term used in the biomedical systems used to impose oxidative stress upon cells have
literature that includes oxygencontaining radicals (such as Of, included exposure to elevated oxygen concentrations
‘OK, RO’, R02’) and non-radical species that can produce oxygcn-
[42], incubation with enzymes that generate reactive ox-
containing radicals during their reactions (HzOz, 03, singlet OrlAg,
HOCI). The term ‘reactive’ is relative; for example, Oi is very much ygen species (such as xanthine oxidase plus its
less chemically reactive than is ‘OH. substrates, xanthine or hypoxanthine [18,22-27]),

Published by Ehevier Sciettce PublMers B. V. 9

rllircct ;additien to the exllxp al H$~z [i8,2&=32,43,44, ~hr~m~%~rn~~I781 tend copper iona &re very cffeetivo in
44e,bf, sPsrgsrnichydr~peroxidem [3%36,44~1lr,45], or of promoting H~~~-d~p~n~l~nt dramage to iaolnted DNA
ceinpound~ whaae metakmlism by the?cell ~eswltr in in= t&7) ana 5~ DNA within chramtstin [71J in vitrs, A x1?-
creased, inrraeeliuirr g~r~~r~ti~n ST Oi and H$& (such. erand ~~$~ibility ir thert ths mcttttl iana might be releasrd
msparaquat tend mcnndieane (LOJI:]}, and c&ncubation within the crll w U nrutt of oxidntive mess, mt then
of the eclls with de-tivrtcd phagacytes such PJ bind to the DNA /71]. Thus, just ns oxidntive stress
mncrephr#cs [38) and neutrophiis [18,39-411, Ar. EBWILC?Lrises in intr~ellula~ free,Clrrc, it rntxy cause rises
civnted ncutrophils and tntlcrophages generate 0: and in intraccllulrr Prcc Iron and&r copper ions that could
HrOp: in addition, +utrephiis produce HOC1 [Zjr46), bind to DNA and make it w target far oxidative dtrmnp
Neutrophils do not produce *OH unleaa a s~urcc OF f72-741, It hna recently been shown chat game chelators
rransition metal ions is added to the incubation mlx- 00 the carcinogenic metal nickel [59] also react with
cure, ix. neutraphils do not them&es appear to can. HZ@&to cause ‘OH-dependent damage to isolated
tain nny form of metnl ion cntalyse that will convert DNA, Mixtures of eobnlt(IIj ions and &Or which arc
HaOr into ‘OH [47-49)‘. Oxidative stress [XJ-541 and thought to produce ‘OH [f!], again damaged DNA in
DNA dnmagc [51J also occur when some mammzGtln a way characicristic of attack by ‘OH (Nzlekerdien,
cclis are exposed to tumor necrosis factor. The DNA Rata, Haliiwcll, and Dizdaroglu, in preparation,
damngt: produced in human cetis by exposure to A second cxphnation of the ability of’oxidativc stress
cigarette smoke [55,55a,56], asbestos [57,58), ozone to CQIISCBNA damage is that it triggers off a series of
(60,61 J or to ecrtain carcinogenic met&, such as nickel metabolic events within the cell 176,771that lead to ac-
(591, haa also been suggested to involve reactive oxygen tivation of nuclcasc enzymes, which cleave the DNA
specks, backbone. There has been muc.h debate recently concer-
ning the suggestion that oxidative stress causes rises in
intracellular free Ca’“, which might fragment DNA by
2. POSSIBLE MECHANISMS OF DNA DAMAGE activating Ca”*-dependent endonucicases [21,37,78] in
INDUCED BY OXIDATIVE STRESS a mechanism resembling that of apoptosis (‘programm-
Why does oxidative st ress cause DNA damage? In the ed cell cieath’), An example of apoptosis is the killing of
case of extcrnaiiy-generated reaccivc oxygen species immature thymocytes by glucocorticoid hormones,
(e.g. when cells arc incubated with HzOZ, activated which activate a self-destructive process that apparently
phagocytes or xanthine oxidase plus its substrates) involves Ca’*-dependent DNA fragmentation [79,80].
damage is usually inhibited, by adding cataiase, showing These two mechanisms (DNA damage by ‘OH or by
that Hz02 is needed. Superoxide dismutase (SOD) does activation of nucleases) arc not mutually exclusive, i.e.
not usually inhibit much, which could mean either that they could both take place. Indeed, there is evidence
Or’ is not involved in the DNA damage, or that SOD consistent with both of them. Their relative importance
does not enter cells easily. That the tatter interpretation may depend on the cell type used and on how the ox-
is correct in at least one cell system is shown by the idative stress is imposed. For example, chelating agents
observations that SOD can protect hepatocytes from that bind iron ions into chelates unable to generate “OH
the toxicity of Hz02 or f-butylhydroperoxide under (such as desferrioxaminc [SlJ, desferrithiocin 1181,or
conditions where it does enter the cells [62,63], phenanthroline [82]) can often protect ceils against
However, neither 0; nor Hz02 undergoes any chemical DNA damage and other toxic effects of oxidative stress
reaction with DNA, as measured by strand breakage [30,31,34,38,73,83-$51, The effects of desfcrrioxamine
[64-661 or by chemical changes in the deoxyribose, are variable, since in general it does not cross cell mem-
purines or pyrimidines [9,67,68]. Hence, DNA damage bran.es readily, although it appears to enter some cell
by oxidative stress cannot involve direct attack of 01 or types (such as hepatocytes) more readily than it enters
of H202 upon the DNA. others. Jonas et al. [29] showed that the toxicity of
Two explanations of the DNA damage have been ad- I-3202to epithelial cells is greatly diminished at 4°C but
vanced (Fig. 1). First, it is possible that the damage is it can be increased again by adding ascorbic acid: this
due to ‘OH radical formation [30]. Thus, it is envisaged effect is not seen if cells are pre-treated with desferriox-
that M202, which crosses biological membranes easily amine. Their observations are consistent with a
[69J, can penetrate to the nucleus and react with ions of mechanism of cell damage that depends on Hz02 and
iron or copper to form ‘OH. Because of the high reac- reduced iron ions: at high temperatures normal
tivity of ‘OH and its resultant inability to diffuse metabolism may provide a reductant (such as OF),
significant distances within the ceil [69], this mechanism tihereas at low temperatures ascorbate can replace it.
is only feasible if the, ‘OH is generated from Hz01 by Suggestive evidence that Fenton-type reactions can oc-
reaction with metal ions bound upon or very close to the cur within bacterial ceils has been presented [87-$93,
DNA. One possibility is that these metal ions might although this is not necessarily relevant to mammalian
always be present bound to the DNA in vivo. For exam- systems. SHydroxyguanine (&OH-Oua) was increased
pie, copper ions are thought to be present in in amount in’the DNA of P388 Dl cells after exposure

10
 Fig. 1, Myporlrcrcs to cxplnin DNA dnmngc resulting from exposing cells to oxihiw StrCs.s.

to I-I202 [18]: d-OH-Gua can arise from attack of ‘OH However, the evidence for metabolic changes pro-
upon guanine (see next section) but should not be pro- duced in cells by oxidative stress is also strong
duced as a result of nuclease action. DNA isolated from [21,39,96-991. Menadione and other quinones (which
P388 Dl cells after exposing them to Hz02 did not show ‘redox cycle’ within cells to give 02 and H202) appear
a regular pattern of fragmentation, such as might be ex- to produce DNA strand breaks in hepatocytes by
pected from nuclease attack [ 181. Increases in g-OH- Ca2+-dependent activation of an endonuclease. DNA
Gua have also been observed in DNA from other cells damage could be inhibited by preventing the rise in
subjected to oxidative stress [90,91]. Thymine glycol, Ca2+ using Ca 2+-chelators [37,94]. Oxidative stress can
another product that can result ,from attack of ‘OH also sometimes activate and/or cause changes in the
upon DNA (see next section) has been reported to be subcellular location of PKC (protein kinase C) [95-993.
formed in the DNA of yeast cells after exposure to high Exposure of mouse epidermal JB6 cells to Hz02 ap-
concentrations of I-I202 [92] and in murine tumor cells peared to cause a Cazc-dependent translocation of PKC
after exposure to tumor necrosis factor [S11. Treatment to the piasma membrane [!X] whereas menadione ac-
of murine hybridoma cells with Hz02 caused a pattern tivated PKC both in these cells and in rat hepatocytes
of chemical changes in the DNA bases that is [95,963 without producing translocation. Cantoni et al.
characteristic of attack by ‘OH [93]. [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 hydroycn btlnda in waFerI then ‘GH can grabrbly bi:
may be nccommodnred by proposing that ohungca in the clnssifitld as a eompizrs carcinogen, Bascqair changes
availability of calcium lona may depend upon, or give and Some frt?rnr&Ma are the eemrnoncot murririsns
risa 10, changer in the availnbility oi+ iron or copper observed in cells exposed to ionizing radiation
ions, Clearly, attempting co elucidate t;tvz mfchanism af (I 10, it I], Chemical ckangcs In the: 5NA bases, aingic-
DNA damage by thr use of free radical scavengers or and double-strand breaks and cnhnnecd cxprcs&on of
metal ion chelators added to the outlsidc of ucllt ix certain prote-oncogcner [&I 12,113] have also been
unlikely 10 give unambiguous answers. Let 11ssee what observed. WOwever, the prceise relationship between
can be learned from the techniques of molecular these differenr events tend rhc dcv~lopmttnt of cancer is
biology and analytical chemistry. 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
3. REACTIVE OXYGEN SPECtES A$ MUTAGENS
error-prone fashion. Resynthesis of BNA after excision
AND CARCINQGENS
repair might conceivably introduce errors.
Oxidative stress, imposed by a variety of mechanisms Thcrt: are many steps between a healthy cell and a
(including increased 02 concentrations [98a]), has been malignant tumor, Cancer biologists have often referred
convincingly shown to be mutagenic to bacteria to at least three stages: initiation (an irreversible change
[69,99-1031. For example, E. coli mutants bxking SOD in DNA), pramotion (probably involving changes in
activity show greatly-enhanced rates OF spontaneous gene cxprcssion) and progression (further changes in
mutation [99]. Similar mutagenic effects have been DNA leading to the cvenrual production of a malignant
shown in a range of mammalian cell types (42,105-1071 tumor). Both Zimmerman and Ccructi [115] and Weitz-
subjected to oxidative stress. Moraes et al. [108] studied man et al. (1161 showed that a clone of C3H mouse
the pattern of mutations obtained in a gene of a shuttle fibroblasts exposed to activated human ncutrophils or
plasmid when simian cells transfectcd with this plasmid to hypoxanthine plus xanthine oxidasc underwent
were exposed to HzOZ. Both single base changes and malignant transformation* Nassi-Calo et al. 1117)
deletions were observed. The majority of base changes showed that Hz02 also transformed these cells, an ac-
were at GC base pairs, the GC-+AT base transition be- tion prevented by the chelating agent o-phenanthrolinr.

Fig. 2. A combined hypothesis. Rises in intracellular free iron or copper ion concentrations could be a consequence of rises in Ca”* (lB, 2B) 01
vice versa (IA, 28).

12
 Radiation chemists have carried we many rrudicP of
Ahhougk mw r\trcnricw haa ken paid in ths the effcct~ of ‘OH, yencrnred by ionisingg radintion,
lircraturr! to I hi(Eacticsn of rewtivf
mypen rpceicr yIx pro- upon DNA, Thix radictt! ia rotarrsctive that it can attack
mw33 of cawcinagencsia [ 115,120,121,123], their ablli- ail csmgoncntx of the DNA (reviewed in (4,138-1403).
ty fo dam~tgc DNA and produce alrerarionu~in gcnc ex= Thus, ‘OH abstracts hydrogen atoms Cam dwx-
prexxion implies that they could bc involved in all srager yribosc, giving su&ar radicals that can fragment in
af careinogeneais [124,127,129]. It bar been argued various ways. Reactions of deoxyribose-derived
[42,130- 132] that canrinuoua: dama~? to DNA by free radicals can lead to lhe release of’ pwine nnd pyrimidine
radical mechanisms ia B significant ERWJCof cftnccr in bnses from the DNA (producing abasic sites), and to
humans, nn abwvation rlw might explain rcaultn of strand breaks. Some of the altered sugars that rrmnin
the epicicmiologiral investigations, which show invlersc artnchcd to DNA can be xplir to give strand breaks by
corrclarions in humans between plasma concentrations incubation with alkaline solutions; these are the so-
of certain an~ioxidants and rhe: incidence of cancer called “alkali-labile sites [4,15,141]. Chemical changes
[ 131,133], However, several anrioxidnnfs can alter the to rhc purine and pyrimidinc bases have also been
metabolism of procarcinagcns, Favoring metabolic characrerized in detail (reviewed in [ 138-140)). Thus,
pathways rhar do nat result in formation of ultimate ‘OH can adcl on to guanine residues at C-4, C-5, and
carcinogens [ 13I ,134]. Menee a protective effect OFan- C-8 positions. For cxamplc, addition OF ‘OH to C-8 OF
tioxidants does 1101r~ecesstrril,~mean rhnt oxidtlrivc yuaninc produces a radical adduet rhat has scvcral
Stress leads fo the cancers in question. Of course, some possible fates. Ir can be reduced to Ghydroxy-7,8-
carcinogens might act by imposing s?n oxidnrive stress dihydroguanine, osidized to &hydroxyyuaninc, or
on their target cells (Section 4), and reactive oxygen undergo ring opening followed by one-electron reduc-
species have been claimed to be capable of converting tion and protonation to give 2,G-diamino-4-hydroxy-S-
some procarcinogcns into ultimate carcinogens formnmidopyrimidinc, usually abbreviated as FapyGua
[134,135]. In addition, it must be borne in mind thar a (Fig8 3), Similarly, ‘OH can add on to C4, CS, or C8 of
high plasma level of such antioxidants as ascorbic acid adcninc residues: among other fates, the C-8 ‘OH
and vitamin E may simply be an index of a good diet, adduct radical can be converted into 8-hydroxyadenine
which protects against many diseases. by oxidation, or undergo ring-opening followed by one-
DNA damage resulting from oxidative stress (or from electron reduction to give 5-formarnido-4,6-diamino-
any other mechanism) need not necessarily lead to pyrimidine (FapyAde). Fig. 4 shows the structures of
cancer. Low lcvcls of damage may be efficiently sonic of these compounds. Pyrimidines in DNA are also
repaired with a minimal risk of error, High levels of ox- attacked to give multiple products. Thus, thymine can
idative stress may lead to cell death, so that initiated form c/s and tram thymine glycols (5,6-dihydroxy-6-
cells do not remain in the organism. Thus, an in- hydrothymines), %hydroxy+methylhydantoin, 5,G-di-
termediate level of damage is most likely to predispose hydrothymine and 5-hydroxymethyluracil. Cytosine
to malignancy, which may explain the close association can form several products, including cytosine glycol
of chronic inflammation (involving phagocytic produc- and 5,Gdihydroxycytosine (Fig. 4).
tion of 02 and H202) with malignancy in such human When whole cells or isolated chrotnatin are exposed
diseases as ulcerative colitis, Crohn’s disease and reflux to ionizing radiation, cross-links can occur between
esophagitis (reviewed in [127]). Cerutti et al. [122] DNA bases and amino acid residues in nuclear proteins
showed that one difference between a clone of mouse [139,142-1481. Thus, thymine-tyrosine [ 1451, thymine-
epiderrnal cells that was promotable by xanthine/xan- aliphatic amino acid [ 143- 1491, and cytosine-tyrosine
thine oxidase and a non-promotable clone was that the [ 1463links have been identified in isolated calf-thymus
latter had lower levels of SOD and catalase and was chromatin subjected to y-irradiation. Treatment of
more sensitive to killing by reactive oxygen species. chromatin with Fe”‘-chelates and Hz02 also produces
Thus, increased antioxidant deferices, by protecting DNA-protein cross-links [153] and such !inkc,have been
against cell death resulting from oxidative stress, might detected in cellular DNA after exposure of the cells to
conceivably (and ironically) sometimes lead to increas- ozone [60].
ed cancer [136,137]. Molecular biologists have examined the likely

13
physiological effects of these various lesions in DNA, situation with other reactive oxygen species is less clear-
GHydrsxyguanine (and, by inference, &hydroxy- cut at prcscnt, Sinylac oxygen is able I0 produce limited
adenine) might lead to mutations by inducing mis- strand breakage in isolated DNA [153,154], and its
reading of the base itself and of the adjacent bases ability co modify the DNA bases is aIso limited (154a].
[llO,ll 1,151,152]. Thymine glycol might have some Thus, M. DizdnrogIu and H. Sits (personal com-
mutagenic action and it can be lethal if not removed munication) found small amounts of d-OH-Gua and
from the DNA before replication [ 110,ll l], Riny- FapyGua but no ocher significant base changes in DNA
fragmcnccd bases are thought co block DNA replication exposed to singlet 0~ gcneratcd by the thermal dccom-
[I 10,ll I]. Abasic sites, which can result from direct at- position of an endopcroxide. Exposure co illuminated
tack of ‘OH, can also be mutagenic in vivo [I 10,l 111, Mechylene blue causes formation of &hydroxyguaninc
It is clear chat ‘OH products multiple changes in [155] and of some strand breaks [I561 in DNA but the
DNA whereas 02” and Hz02 have no effect, but the species responsible was not identified, except for the

S-hydroxy-fi-mthyl- S-hydroryhydmtofn 5Aydroxyurocil

hydmtoin

S-hydtoxymotbyl- S-hydroxycytoslne thymine elycol 5,6-dihydcoxy- 5,6-dihydroxy-

uracil uracil cytosinc

4,G-diamLm-5- 8-hydroxyadcnino 2,6-dlamfno-4-hydroxy- 8-hydroxygunnine

Pormmidopyr@ddina 5-fomiamidopyrimidinc

Fig. 4, Some of the end-products that result from attack of hydroxyl radicals upon the bases of’DNA.

14
Enbiefvationthar rcavenflarr at ‘OH did not protccc. xyd~o~~~~~~n~~~n~ huve bet?n detected in rn~rnrn~t~~n
Hawcver,dha excited state of the ph~to~en~iti~~n~ ,dya urine [f35,175-L781. The prcwxe erf these produan In
Raw&2bengIl hWKiwlf been clrSmed ta clewve DNA [ 157) urine ~~~~~e~r~that axidativc damage to t hc fS‘NA bwec~
and, if ‘OH were $anerurcd by Mcthyirne blue bound to daes cxcuf In viva, and thwr repair nyatcnx ure wetiw to
the DNA, then ‘01-I srcavenfi~r%
would nar b+ experfcd cleave modified b~tscir from DNA, ~“~OVI”CVC~, it ia pnnsi-
10 pmteef. Elluminated ribcafhwin, which aenrratca tale! that xomc excreted bases ori&ate from the dicr or
rinatet CL WWJ regarted no1 to pradusc I-hydrtlxy- from the mernbralism af rho gut flora, and chat DNA
gurninc in DNA [l%t]. Thus, sin@et 8: certainly does relewd fram dead and dying cells within an erganlsm
nat induce the extenrive pattern af DNA base modifieta* undergo+% rcyitl oxidaticr drmaae (since cell disruption
tion produced by ‘a-s. can incrensc Free radical reaetions [69,72,164]). Hence,
Peroxidixing lipids hwe been reported to dxlmnp one must be eaurious in using the amounts af modified
DNA flS9=163a] but peroxidizing lipids produce a DNA brracaexcreted ftam the body tls an index of thr
rengc of reactive oxygen species including ‘OH, W&II, extent of repair of oxidative DNA damage in hartlrlr~
singlet oxyycn, peroxyl radicnls, and nlkoxyl radicals eclls.
[69,164] i0xl the exact contributions of these species to
the DNA damage observed need to be determined
5. MEASUREMENT OF BASE-DERW33
[165,166]. Lipid peroxides alno decompose to give tl
PKXGJCTS AS A PROBE FOR THE
huge range of producta [166-1683 ineluding carbonyl
MECHANISM AND EXTENT OF DNA
compounds, such na maiondia~dehyde and the un-
DAMAGE
saturated tlldchyde 4-hydroxy*2-lrnrrs nonenal [ 168]
which has been shown to be mutagenic to mammalian The development of an I-WK tcchniquc, coupled
cells [ 1691. If these aldehydcs arc generated in the vicini- with highly-sensitive clcetroehemical detection, for the
ty of DNA, they mlry be able to combine with it to form measurement of &hydroxy-deoxyguanosine has led to a
distinctive products [ 165,169], Thus, malondialdchydc series of pioneering studies in which measurement aF
reacts with adcnine, eytosine, and guaninc [161,17011] this product has been used to gain information about
and a guaninc-MDA adduet has been identified in free eadical damage to DNA in intact cells and whole
human urine [ 1711,The product of reaction of hydroxy- organisms [91,114,179-1811. Antibody techniques for
nonenal with deoxyguanosinc has also been characceriz- the measurement of such products as &hydroxyguanine
ed [172]. and thymine glycol have also long been available (e.g.
Humans arc constantly exposed to background levels see [182-184)). Ames et al. [175-1771 have used the
of ionizing radiation, which will generate some ‘OH in urinary excretion of products derived from guaninc and
vivo. This radical may also arise by reaction of metal thyminc as an index of free radical damage to DNA in
ions with H;rOzin vivo [5]. Thus, it is not surprising to vivo, and have attempted to draw conclusions about
find that repair systems have evolved to remove at least changes in the rate of such damage as a function of age
sotne of the lesions in DNA that can result from attack in mammals. The amount of 8-hydroxyguanine in the
of ‘OH and other reactive oxygen species (reviewed in DNA from certain sub-populations of eat liver
[ 173,174]). Single-strand breaks, ate usually quickly mitochondria was found to be considerably higher than
repaired; indeed, they are generated as intermediates in that in nuclear DNA, leading to proposals about the
the repair of other lesions (see below). 8-Hydtoxy- role of mitochondria in aging and carcinogenesis
guanine is slowly removed from cellular DNA, but the [ 185,186]. Exposure of some cells to oxidative stress has
repair mechanism is unknown [ 1731.Several lesions, in- been reported to lead to formation of Wydroxy-
cluding thymine glycol, are probably removed in guanine in the DNA [22,90,91 ,114]. For example, treat-
human cells by action of a DNA glycosylase, which cuts ment of Ehrlich ascites cells with the carcinogen
the base-deoxyribose bond to give an abasic site. This 4-nitroquinoline l-oxide led to an increased content of
site is recognized by an endonuclease activity (on the 8-hydroxyguanine in DNA [187]. Intra-peritoneal injec-
same enzyme), which nicks the strand at the abasic site. tion of ferric-nitrilotriacetic acid (which reacts with
The damaged part of the strand is removed, followed by HZOZto give ‘OH [9,10]) into rats produced a signifi-
resynthesis of the DNA and re-joining of the strand by cant rise in the %hydroxyguanine content of kidney
a DNA ligase enzyme. Glycosylases that recognize DNA [lSSJ. Carcinogenic peroxisome proliferators
hydroxymethyluracil and ring-opened purines in DNA [189,190], acetoxime [191], 2-nitropropane [191] and,
have also been described in mammalian cells in one study, a choline-deficient diet [lgla], have all
[110,111,173,1743. been reported to result in increased amounts of
Modified DNA bases and nucleosides (base-deoxy- 8-hydroxyguanine in DNA in vivo in mammals.
ribose) have been detected in the urine of humans and These studies have certainly produced qualitative
other mammals. Thus, S-hydroxyadenine, 7-methyl-8 evidence for oxidative damage to DNA in vivo,
-hydroxyguanine, thymine glycol, thymidine glycol, although care must be used in interpreting the data
hydroxymethylutacil, 8-hydroxyguanine and B-hydao- [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 rcac-
rive 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 cx-
ample), 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 0.55 0.57
substantial fortnarion of FnpyGua as well as I,8 3.5
84tydroxyguaninc in DNA (671, whereas systems con- 0.8 0.95
taining copper ions and Hz01 greatly favored 3.4 4.5
8-hydroxyguaninc production [6,7 I, 1931. When
isolated, mammalian chromatin was irradiated in
aqueous suspension, the relative atnounts of 8-hydro-
xypurines and formamidopyrimidines generated
depended upon the radical environment provided by the nucleases should leave the purines and pyrimidines
gases used to saturate the aqueous solution [194]. For unaltered (Figs. 1 and 2). For studies on DNA
example, the presence of oxygen favoured the forma- modification, extraction of chromatin from cells for
tion of %hydroxypurines [19$196]. Table I summarizes analysis is preferable to extraction of DNA, since it
some of rhc results obtained, Products derived from minimizes the loss of extensively-fragmented DNA, and
pyrimidincs can similarly bc affected by changes in of DNA that has becotne covalently cross-linked fo pro-
reaction conditions [195,196]. tein.
A complete characterization of damage to DNA by GUMS-SIM has been used to characterize the
reactive oxygen species can be achicv,ed bg tl;; tcchni- damage done to DNA by various reactive oxygen
que of gas chromatography/mass spectrometry (review- species. Hydroxyl radical appears to produce a
ed in [194a,197,198]), which may be applied to DNA uniquely-extensive pattern of base modifications
itself or to DNA-protein complexes such as chromatin. (multiple products from all four bases), cind this pattern
The DNA or chromatin are hydrolyzed and the pro- seems to be a ‘fingerprint’ for ‘OH, i.e., it can be used
ducts .converted to volatile derivatives, which are to identify ‘OH as a damaging species [6,9,68,
separated by gas chromatography and identified by 71,139,143,146,152,193,194,196,198,201]. For exam-
mass spectromctry. High sensitivity of detection can be ple, measurement of damage to the DNA bases by
achieved by operating the mass spectrometer in the GUMS-SIM has been used to show that the strand
selected ion monitoring (SIM) mode. In this mode, the cleavage produced in isolated DNA by treatment with a
mass spectrometer is set to monitor several ions derived copper-ion phenanthroline &elate probably involves
by fragmentation of a particular product during the ‘OH [ 1931,whereas DNA cleavage by a bleomycin-iron
time at which this product is expected to emerge from ion complex is not mediated by ‘OH [ 1991.GUMS has
the GC column. The GUMS-SIM technique is being also been used to identify &OH&a, FApyAde,
used in the authors’ laboratories to examine the 8-OHAde and FApyGua in neoplastic tissues [200], to
mechanism by which DNA is damaged in cells subjected show that the damage done to the bases in isolated
to oxidative stress. Thus, if damage is due to ‘OH DNA by activated human neutrophils is most likely due
generation, then products characteristic of ‘OH attack to ‘OH generated by reactions involving metal ions in
should be detected (Fig. I), as has been observed in the reaction mixture [2Ol], to measure adducts of car-
murine hybridoma cells treated with Hz02 [93] and in cinogens with proteins in vivo in attempts to assess car-
primate trachea1 epithelial cells exposed to ozone cinogen exposure [202] and to characterize the changes
(Aruoma, Halliwell and Wu, in preparation). By con- produced in plasmid DNA by treating it with potassium
trast, cleavage of the DNA backbone by the action of permanganate [203]. The authors believe that such

16
 Dypbukt, J,M., Thor, H. nnd Nirtlldrn, P. (I99Oj Prcc
Rrrtlirttl Rcs, Carn~~~ur~,6, 34&&W,
Chang, Y&Z., Hcppner, G.H., RIIII, L.A. and ~&on. A.M.
(1989) Canfcr Rer. 49, 6652-6657,
REFERENCES Weilher&, A.W,, Wllxnran, S,A., Desrrempes, M,, but, S.A.
[I] Frictsvictr, 1. (1989) Annu. Rev, Pharrn, ‘l’us. 23, 239-257. and Smrxrl, TSP. (1983) New En& J. Med. 308, 26-29.
[ZJ lies, M,, ed. (1991) Oxida~ivc Stress: Oxitlttntn nntl W&berg, A.B., Writamrn, $.A., Clark, E.P. tend Srernci,
Antlo?iichtntr, Aeadenrie Press, New Yurk. T.P, (1985;) J, Clin. Inwt, 75, 1835-1141.
[3] Weiss, S.J. (1989) New Engl. J. Med. 320, 365-376. Shncler, E., Bcccham, E.J,, Covey, J.M., K&n, l&W, nnd
[4) Van Sonnt:& C. (1417) The Chemienl U;rris of Rndiarlon Porter, M* (1988) Ctrrcino~enrais 9, 2297-23(&l,
13iotoayl Tnylar and Francis, London. Gille, J,J,P,, Mullnart, E,, Vijg, J., beyrr, A-b,, Arwcrt, P.
[5) Helliwell, B. and Gutrcridpi, J#M,C. (1990) Me~hath nnd Joenje, M, (1989) Murnt. Rex. 219, 17-28.
Enrynrol, 86, I-l& Cantoni, a,, Cattabeni, E., Stocchi, V,, Meyn, i&E,, Cerutti,
(6) Arrmmn, G.I., Malliwell, B,, Gejcwski, E. and Dirduraylu, P. ond Murray, D. (1989) Biochim. Biophys. Aeta 1014, l-7,
M. (1991) Biachcm, J., 273, 601-604, Prisc, K.M., Davies, S. and Michael, B.D. (1989) Inc. 1. Rad.
(71 Sagripunti. J.L. and Kramer, K,W. (1984) J. Riot. Chrnr. 264, Bid, SS, 583-592.
1729-1734. Snnrlrom, B,E. and M~rklund, S,L. (1990) Bioehem. J. 271,
18) Inoue, S. nnd Knwnnixhi, S, {1987) Cancer Rcs. 47, 17-23.
6522-6527. [44b] Spragg, R.G. (1991) Am. J, Rcspir. Cell. Mot, Biol. 4, J-IO.
[9] Aruarns, O,l., ~Ialliwril, I~,, Cajewaki, E. niitl t)izciaraglu, 1451 Nicotcrn, P., McConkcy, D., Svensron, S.A., Beitomo, 6.
M. (1989) J, Biol. Chem. 264, 20509-20512. and Ortcnius, S. (1988) Toxicology 52, 55-63,
[IO] GutleridBe, J.M.C. (1990) Free Radical Rcs. Co~~~mun. 9, ,I461. Curnuttc, J.T, and Bnbior. B,M, (19871. . Adv. Human Gcaet.
I lO-125. 6, 229-247.
(111 Halliwcli, B. mrtl Gutterid&, J,M.C. (1990) Arch, Biochem. [47] Thomas, P.J., Shirley, P.S,, Hedrick, CC. and De Chntelct,
Biophys, 280, I-8. L.R. (1986) Biochemistry 25, 8042-8048.
1121 Wayner, D,D.M., Burton, G.W. nnd Inyold, K.U. (1986) [48] Uritigan, BE., Coffmitn, T.J. and Buettner, O.K. (1990) J.
Biochim. Biophys. Acta 884, 119-123. Biol, Chenr. 265, 2650-2656.
1131 Frei, l3., England, L. and ,Amcs, B,N. (1989) Proc. Nari, 1491 Kaur, H., Fagerheim, I., Grootveid, M., Puppo, A, nnd
Acad. Sri. USA 86, 6377-6381, Waiiiwell. B. (1988) Anal. Biochem. 172. 360-367.
(141 Birnboim, H.C, (1990) Methods Enzymol. 186, 550-555, [50] Larrick, ‘J.W‘. and Wright, S,C, (li90) FASEB .I. 4,
iI51 Ahnstrdm, G. (1988) Int, J. Radiat. Bioi. 54, 695-707. 3215-3223.
(161 Bhusatc, L.L., Herbert, K.E. and Perrctt, D. (1990) Biochcm, [Sl] Zimmerman, R.J,, Ghan, A. and Leadon, S.A. (1967) Cancer
Sot. Trans. 18, G7G-677. Res. 49, 1644-1648.
iI71 Schraufstatlet, I.U., Hinshnw, D.B., Hyslop, P.A., Spragg, [52] Won& G. end Goeddel, D. (1986) Science 242, 941-944.
R.Ci. and Cochrane, C.G. (1986) J, Clin, lnvcst. 77, [53] Shaffer, J.B,, Treanor, C.P, and Del Vecchio, P.J. (1990)
1312-1320. Free Rocl. Biol. Med. 8, 497-502,
iI81 Schraufstatter, I.U., Hyslop, P.A., Jackson, J.H. and [54] Hauser, G,J., McIntosh, J.K., Travis, W.D. and Rosenbery,
Cochrane. C.G. (1988) J. Ciin, Invest. 82. 1040-1050. S,A. (1990) Cancer Res. 50, 3503-3508.
[I91 Eklow-La&orn,‘L., kossi, L,, Thor, Hi and Orrenius, S, [SS] Borish, E.T,, Cosgrovc, J.P., Church, D.F., Dcutsch, W.A.
(1986) Free Radical Res. Cornrnun. 2, 57-68. and Pryor, W.A, (1985) Biochem. Biophys. Res. Commun.
WI Starke, P.E., Hock, J.B. and Farber, I.L, (1986) J, Biol. 133, 780-786.
Chem, 261, 3006-3012. [55al Kiyosawa, H., Suko, M., Okudaira, H., Murata, K.,
PI1 Orrenius, S., McConkey, D.J., Bcllomo, G, and Nicotera, P. Miyamoto, T., Chung, M,H., Kasai, H. and Nishimura, S.
(1989) Trends Pharm. Sci. 10, 281-285. I1990) Free Radical Res. Commun. 11. 23-27.
PI W&berg, A.B. (1989) Mutat. Rcs. 216, 197-201. [56] Nakajama, T., Kaneko, M,, Kodama, M. and Nagata, C.
P31 Paul, L.A., Fulton, A.M. and Heppncr, G,H. (1989) Mutat. (1985) Nature 314, 462-464.
Res, 215, 223-234. [57] Weitzman, S.A. and Graceffa, P. (1984) Arch. Biochem.
~241 c‘rhillips, B.J., James, T.E.B. and Anderson, D. (1984) Mutat. Biophys. 228, 373-376.
Res. 126, 2G5-271. [SS] Jackson, J.H., Schraufstatter, I.U., Hyslop, P.A., Vosbeck,
[251 lwata, K., Shibuya, H,, Ohkawa, Y, and Inui, N. (1984) K.. Sauerheber. R.. Weitzman. S.A. and Cochrane. C.G.
Tonicol. Lett. 22, 75=51. (2987) JI Clif?, mnve;t< 80, 1999-109s..
[26] Hall, A.H. Jr., Eanes, R.Z., Waymack, P.P., Jr. and [59] Kawanishi, S,, Inoue, S. and Yamamoto, K. (1989)
Patterson, R,M. (1988) Mutat. Res. 198, 161-168. Carcinogcnesis 10, 2231-2239.
[271 Carson, D.A., Seto, S. and Wasson, D.B. (1986) J. Exp. Med. [60] Van der Zee, J., Van Beck, E., Dubbelman, T.M.A.R. and
163, 746-751. Van Steveninck, J. (1987) Biochem. J. 247, 69-72.
WI Olson, M.J. (1988) J. Tox. Environ. With. 23, 407-423. [61] Borek, C., Zaider, M., Ong, A., Mason, H. and Whz, G.
1291 Jonas, SK,, Riley, PA. and Wilison, R,L. (1989) Biochem. (1986) Carcinogenesis 7, 1611-1613.
,LT
J. 264, 651-655. II
[Qs] Brawn, M.K. #nd Fridovlch, I. tl9@4l) Arch, Isioehilm.
Diophyr. 286, JIG41%
[&$I Le~ko, %A,, Lorrnr~zn, R.J. ltnd Tsu, P O.P. (l%tl,
t3iathcmi5lry IQ, 3#23-3628,
166) Rowlcy, 4X&_, and H~lliwcll. 43. (IQ831 43iucbim. Biaphys,
hfln 761, M-!a.
(67) Aruoma, O,I., Halliwcll, H. and l34xdaropht, M. (i.089) J.
Biol. Chcm. 71sL, 13023-13628.
(68) Blukely, W-F., +uclarclli, h.F>, Wcgher, 4&J, and
Dizdnruglu, kl, (199O) Radirl, Rex, 121, 33X-343. [ IOj] Abril, N. and Puoyo, C, (l99O)Envlron, Molce, Mu~ugen. IS,
[69) Wnlliwc?ll, 13, and Ciutleridpo, J.M.C. (4989) Prcc Ratlic~\lr in 184-189.
Biology and hlcdicinc, second crlition, Clr\rcl~tlon PrK% [lU5] Wcitxman, S.A. and S~usuel, T-P. (1981) Science 212,
Oxford, 546-548,
170) Prutx, W.h,, l3utlcr, 3, and Land, E.J. (1990) inr. J, RatI, [IW) W&man, S.A. and Sroxicl, T.P. (1984) Cnncer tort. 22,
Biol. 58, 215-234. 337-342.
(71) Dizdnroglu, M., Rae, O., Hnlliwell, R. and Cinjcwrki, E. [IO71 Elxic, A,Wsr Rcclo, L., ICar& D.S,, Leo, C.Q., Wttgncr, M.
(1991) Arch. Biochcm, Biophys. 285, 317-324, nnd Sehcnlcy, R,L. (19863 Pror’. Nnrl, Acrid. Scl, USA 83,
172) Hnlliwell, 13, (19#7) FASEU J. I, X8-364, %I 6-9620.
(73) Snkaidn, I., Kyle. ME, and Farber, J-L, (199[I) Mol. [IO81 Marncs, E.C., Kcyrc, S.M, anti fyrrell, R.M. (1990)
Pharmacol. 37, 435+2. Crreinogrncdis II, 283-203.
(741 Strrkc, P.E., Gilbertson, J.D, and Fnrbrr. J.L. (1985) [IO91 Moracs, EC,, Keyac, S.M., Pidoua, M.’ and Tyrrcll, R.M.
Biochem. Biophyr, Res. Commun, 133, 37 l-379. (1989) Nucleic Arid% Rcs. 17, 8301-8312.
[75) Moorhouse, C.P,, Halliwell, B,, Grootveld, M. and [I IO] Brrimer, L-H. [1988) Br. J, Cancer 57, 6-18.
Guttcridge, J.M.C. (1985) Biochim. Biophyx. ACIO 843, [Ill] Brcimer, L-H. (1990) Mol. Cnrcinogencsis 3, 188-137.
261-268, 1112) %icgler-Skylnkrkir, K. and Andrea, U. (I987) Murat. Rcr.
[76] Birnboim, H.C. and Kanabus.Kaminskn, M. (1985) Proc. 192, 65-67.
Nati. Acad. Ssi. USA 82, 6820-6824. [I 13) Yamashina, K,, Miller, B, and Heppncr, O.H. (1986) Cancer
[77] Dirnboim, W.C. (1988) Rioshem. Cell Biol. 66, 374-381. Rcs, 46, 23Y6-2400.
[78) Farber, J-L, (1990) Chcm. Rcs, Tax. 3, 503-508. [I 141 Floyd, R.A. (1990) Carcinogenesis I I, 1447-1450.
[78n] Orrenius, S,, McConkey, D.J., Jones, D.P. and Nicotem, P. [I IS) Zimmermnn, R. and Cerurti, P. (l9R4) Proc. Nn~l. Acad, Sci,
(1988) ISI Atlas Sci. Phnrmncoi. I, 319-324. USA 81. 2085-2087,
[79] McConkcy, D-J,, HartzelI, P., Nicotcra, P. and Orrenius, S, (II61 Wcilzman, S.A. Weitberg, AU., Clark, E.P, and S~osscl,
(1989) FASEB J. 3, 1843-1849. T.P, (1985) Science 227, 1231-1233.
[80) Arends, M,J., Morris, R.G. and Wyllic, A-H. (1990) Am, J. [I 17) NassXalo, L,, Meilo-Fllho. A.C, and Meneghini, R. (1989)
Path. 136, 593-608, Carcinogencsis IO, 105%1057,
[Sl] Haiiiwell, B, (1989) Free Rad. Biol. Med, 7, 645-651, [I 18) Weitberg, A.B. and Corvese, D. (1990) Biochem. Uiophys.
1821 Halliweil, B. (1978) FEBS Lett. 96, 238-242. Rcs, Commun, i69,70-73.
[83] Meneghini, R. (1988) Mutat. Res. 195, 215-230. [I 191 Radosevlch, CA. and Wcitzmnn, S.A. (1989) Carcinogcnesis
[84] Kyle, M.E., Nakac, D., Sakaida, I., Serroni, A. and Farber, 10, 1943-1946.
J.L. (1989) Biochem, Pharmacol. 38, 3797-3805. [I201 Sherman, M-L., Dalta, R., Hallahan, D.E., Weichsclbaum,
[SS] Starke, P.E. and Farber, J.L. (1985) J. Biol, Chcm. 260, R.R. and Kufe, D.W, (1990) Proc. Nati. Acad. Sci. USA 87,’
10099-10104, 5663-5666,
[86] Gannon, D,E., Varaai, J., Phan, S.H., Ward, ,I,H, ( Kapinn, [I211 Shibanumn, WI., Kuroki,T. and Nose, K. (1988) Oncogcne 3,
J., Till, G,O., Simon, R.H ,, Ryan, US. and Ward, P.A. 17-21.
119871 Lab. Invest. 57. 37-44. [1221Cerutti, P., Larsson, R., Krupitra, G., Muehlmatter, D.,
[871 imla;, J,A, and Linn,.S. (1988) Science 240, 1302-1309. Crawford, D. and Amstad, P, (1989) Mutar. Res, 214, 81-88.
[881 Brandi,G., Cattabeni, F., Aibano, A. and Cantoni, 0. (1989) (1231 Cerutti, P. (1985) Science 227. 375-381.
Free Radical Res, Commun. 6, 47-55. 11241 Perchelctt, J.P. and Perchelle;, E.M. (1989) Free Rad. Biol.
[W Hassett, D,J,, Bean, K., Biswas, G. and Cohen, MS. (1989) Med. 7. 377-408.
Free Radical Res, Commun. 7, 83-87. [I251 Kensler., T.W. and Taffe, B,G. (1986) Adv. Free Rad. Biol.
[901 Floyd, R.A., Watson, J.J., Harris, J., West, M. and Wong, Med. 2.347-387.
P.K. (1986) Biochem. Biophys, Res. Commun. 137.841-846, [I261 Goldstein, B,D,, Cecrniecki, B. and Witz, C, (1989) Environ.
[9Il Kasai, H., Grain, P-F,, Kuchino, Y ., Nishimura, S., Health Perspect. 81, 55-57.
Ootsuyama, A. and Tanooka, H. (1986) Carcinogenesis 7, [I271 Weitzman, S.A. and Gordon, L.I. (1990) Bloc3 76, G55-663.
1849-1851, [I281 Halliwell, B, and Gutteridge, J.M.C. (1989) Free Radicals in
[921 Kaneko, M., Leadon, §.A. and lto, T. (1988) Mutat. Res. Biology and Medicine, Second Edition, Clarendon Press,
207, 17-22. Oxford, Chapter 8.
I931 Dizdaroglu, M., Nackerdien, Z., Chao, B.C., Gajewski, E. [I291 Stich, H.F, and Anders, 1;. (1989) Mutat, Res. 214, 47-61,
and Rao, G. (1991) Arch. Biochem. Biophys, 285, 388-390, [I301 Totter, J.R. (1980) Proc. Nati. Acad. Sci. USA 77,
1941 McConkey, D.J., Iiartzell, P., Nicotera, P., Wyllic, AXI+ 1763-1167,
and Orrenius, S. (1988) Toxicol. Lett. 42, 123-130. [I311 Ames, B.N. (1983) Science 221, 1256-1264.
[951 Larsson, R. and Cerutti, P. (1989) Cancer Res. 49,5627-5632. [1321 Ames, B.N. (1989) Free Radical Res. Commun. 7, 121-128.
I961 Kass, G&N., Duddy, S.K. and Orrenius, S. (1989) Biochem. [I331 Gcy, K.F., Stahelia, H.B. and Brubacher, G.B. (1988) in:
J. 260, 499-507. Medical, Biochemical and Chemical Aspects of Free Radicals.
I971 Gopalakrishna, R. and Anderson, W.B. (1989) Proc. Naci. In Proceedings of the 4th Biennial General Meeting of the
Acad. Sci, USA 86,6758-6762. Society for Free Radical Research (Hayaishi, O., Niki, E.,
18 Kondo, M, and Yoshikawa, T. eds,) Elsevier, Amsterdam,
pp, 371-384.
 Wiley&lnr, F&w York, pp. lO9- I15,
I’175) Carhenrr, R,,Sshwicrs, E., Saul, RI.,. and Ames, B,N. Cl91)U
Pruc, Narl, &ad. !%I. USA tit (,5633-5637.
I 176) Adclmnn, R., Saul, R.L. and Amcr, #,N. (IBM) Proc, MuI.
4snd. Set, USA 85, 27&I-2701.
177) Frngs. C.C., Shl#cnrkn, M.&L, Park, J.W,, Dugan, P. antl
3463-3467. Ames, I#&, (lY%l) Pro& &\I. Acad. Ssi. USA 87,
[l4b] I>ixdaru$hi, M., Cjajowski, E., Rcddy, P, and M;\ryolin, S.A. 4533-4537,
(I9891 Blurhrmi~rrr 58. 3625-3621, L178) Stillwell, W-6.. XII, 14,X., Atlkinr, J.A,. Wirlrnok, .l.S. turd
fl46) Bajewrkl, E. and ‘Bixtlaroglu, M. (1990) Uldchcmislry 29, ‘Pu1mcn1wn1, S.K. (lYtI9) Chern. Rex. Toxieol. 2, 94-99‘
977-9!xl. [ I791 Floyd, R. A., Watson, J. J., Wang, P.K., Allmiller, D.H, and
(147) Cress, A.@. nnd Uowdcn, a-r. (1913) Radiar. Rex. 9% Rieknrcl, 8.6. (1916) &cc Ratlisrl Rcs, Commrrn. I, 163-171.
610-4111. [I%01 Floyd, RaA,, West, MS,, EneIf, K,I.., Schneider, J.E.,
[IJlt] Oleinick, N,L,, Chiu, S., Ramakrlxhman, N. nntl Xuc, I+. Won& PX., Tinpcy, D.T, and WB~ICII, W.E, (1990) Anill.
(1987) Rr. J, Cancer 55 (suppl. Vlll), 135-140. Riochcm. 1118, 1%IS&l.
[I491 Gajewskt, E., Fucirrclli, E.F, tmtl Dizdaroglu, M, (1988) Inr, [IHIll Floyd, R.h,, Wcsr, MS., Hopscu, W.Q. and Tin&q. D.T.
J. Hwlia~, 8iol. 54, 555-45’1. (1989) Ptonr Physiol. 91, 644-647,
[I501 tcxko, S,O., Drocourr, J.L. rrnd Yang, S.U, (1982) [I821 Kastri, I+-luid Nishimura, S, (1986) Environ, Wealth Pcrxpccr.
Biocheminry 21, JOlO-5QIS. 67, Ili-El&
tl511
- . Kuchino, Y,, Mori, F., Kasai, H,, Inouc, H., Iwai, S,, Miura, [I831 Kancko, M. and Leadon, %A, (1986) Cancer Rcr, 46, 71-75.
K., Ohtruka, E. und Nishimure, S, (1987) Nature 327, 77-79. [l14] Hubbard, M., Hunng, H., Lnapia, M.F., Idc, H., Erlrngtr,
(I521 Wood, M,L,, Dizdaroglu, M., Gnjcwski, E. and Essigmann, l&F, and Wsllace, S.S, (1989) Radiat. Rsr. 118, 257-268,
J.M+ (1990) F3iochcmiwrry 29, 7024-7032. [I851 Richter, C., Park, S,W.-and Amrr,,B.N. (I%%) From. Narl.
[ISJ] Di Mnscio, P,, Wercrs, H., Do-Thi, H.P.. Laflcur, M.V,M. Acad, Scl, USA 85, 6465-6467.
and Sits. H, (19891 Biochim. Biophyx. Acre 1007, 151-157. [ISd] Richter, C. (1988) FEBS Lets, 241, l-5.
Blarck, e,R,, Peak, J.G, and P&k,-M-J, (1989) Pholochcm. (I871 Kohda, K,, Tada, M., Kasai, H., Nishimura,S. and Kawazoc,
Phorobiol. 49, 607-613, Y. (1986) Biochem. Biophys. Rcs, Commun, 139, 626-632.
Mullcr, E,, Boitmux, S., Cunningham, R,P, and Epc, B. IlW Umemura. T.. Sai. K,. Takani, A,. Hascgawa, R. and
(1990) Nucleic Acids Rcs. I& 5969-5973. Kurokawa, Y, ‘( 1996) C&cinoge&sir I iI 3451147.
Schneider, J.E,, Price, S., Maids, L., Gultcrldge, J.M,C. ~hnd II891 Kasai, H,, Okada, Y,. Nishimura, S., Rao, H.S. and Rcddy,
Floyd, R.A. (1990) Nucleic Acids Res. 18, 631-635. J.K. (1989) Cancer Rcs. 49, 2603-2605.
Epc, B., Hcglcr, J. and Wild, D, (1989) Cnrcinogenesis IO, [I901 Hussain, NS,, Conaway, C.C., Guo, N., Asaad, W. and
2039-2024. Fiala, E-S. (1990) Carcinogencsis II, lOl3-1016.
Peak, J,M., Peak, J.G., Foote, C.S. and Krinsky, N-1. (1984) I1911 Takagi, A., Sai, K., Umcmura, T., Hascgawa, R. and
J. Photoclw~~~. 25, 309-315. Kurokawn, Y. (1990) Jpn. J, Cancer Rcs. 81, 213-215.
Floyd, R,A,, West, M,S.,“Eneff, K.L. and Schneider, J,E. [19la] Hinrichsen, L.I., Floyd, R,A, and Suditovsky, 0, (1990)
(1990) Free Rad. Viol. Med. 8, 327-330, Carcinogcncsis I I, 1879-1881.
Hrambilln, G,, Martelli, A. and Mnrinari, U.M. (1989) Mutat. (19lbj Hegi, ME., Ulrich, D., Sagclrdorf, P., Richter, C. and I.u&
Res. 214, 123-127, W.K. (1990) Mutat. Res. 238, 325-329.
II601 Kasai, H. and Nishimura, S. (1988) in: Medical, Biochemical ~1921 Kobda, K., Tada, M., Hakura, A., Kasai, H. and Kawazoe,
and Chemical Aspects of Free Radicals (Hay&hi, O., Niki, Y. 119871 Biochern. Bionhvs Res. Commun. 149. 1141-l 148,
E., Kondo, M. and Qoshikawa, T. eds.)Elscvier, Amsterdam, [l931 Dizharo$tu, M., Antom& 0.1. and Halliweli, B. (1990)
1021-1023. Biochemistry 29, 8447-845 1,
I1611 Vaca, C.E., Wilhelm, J. and Harms.Ringdahl, M. (1988) [194a] Dizdaroylu, M. and Gajewski, E. (1990) Methods Enzymol.
Mutat. Res, 195, 137-149, 186, 530-544.
[I621 lnouy, S. (1984) FEBS Lett. 172, 231-234. [1951 Tofigh, S. and Frenkel, K. (1989) Free Rad. Biol. Med, 7,
[I631 Hruszkcwycz, A,M. and Bertgold, D.S. (1990) Mut. Res. 244, 131-143.
123-128. [I961 Fuciarclti, A.F., Wegher, B,J., Blakcly, W.F. and
[163a) Hruszkewcwycz. A.M. (1988) Biochcm, Biophys. Res. Dizdaroglu, M. (1990) Int. J. Radiat. Biot. 58, 397-415,
Commun. 153, 191-197. [I971 Dizdaroglu, M. (1985) Anal. Biochcm. 144, 593-603.
I1641 Gutteridge, J.M ,C. and Halliwelt, B. (1990) Trends Biochem. 11981 Dizdaroglu, M. (1991) Free Rad, Biol. Med., in press.
Sci. IS, 129-135. [I991 Gajewski, E., A.ruoma, O.I., Dizdarogtu, M. and Halliwell,
[I651 Morita, .I., Veda, K,, Nakai, K., Baba, Y. and Komano, T. B. (1991) Biochemistry, in press.
(1983) Agric, Biol. Chem. 47, 2977-2979, [2001 Malins. DC.. Ostrander. O.K.. Haimanot. R. and Williams.
[166] Frankel, E.N., Neff, W.E., Brooks, D.D. and Fujimoto, K. P. (19jO) Car&ogenesib 11, 1045-1047.
(1987) Biochim. Biophys, Acta 919, 239-244. [2Oll Jackson, J.H., Gajcwski, E., Schraufstatter, I.U., Hyslop,
[I671 Frankel, E.N. (1987) Chem. Phys. Lipids 44, 73-85. P.A., Furiaretli, A.F.. Cochrane, C.G. and Dizdaroglo, M.
[I681 Esterbaucr, H., Zollner, H. and Schaur, R.J. (1988) ISI Atlas (1989) J. Clin. Invest. 84, 1644-1649.
Sci. Biochem. I, 311-317. [202] Skipper, P.L. and Tannenbaum, S.R. (1990) Carcinogenesis
[169] Brambilla, G., Sciaba, L,, Faggin, P., Maura, A., Marinari, 11, 507-518.
U.M., Ferro, M. andEsterbauer, H. (1986) Mutat. Res. 171, [203] Akman, S.A., Doroshow, J.H. and Dizdarogiu, M. (1990)
169-176. Arch. Biochem. Biophys. 282, 202-205.
[I701 Stone, K., Ksebati, M.B. and Marnett, L.J. (1990) Chem. 19
Res. Tox. 3, 33-38.