Volume 309. number 2, 193-198 FEES 11503 0 1992 Fcdcroltion of Europclln Biochemical Socic~icr 00145733IWWX1
Scptcmhr 1992
DNA base modifications in ckromatin of human cancerous tissues Ryszard OlinskPb, ‘f’omasz Zastawnyb, Jsroslaw Budzbon”, Jan Skokowskib,Wojtck Zqarskib and Miral Dizdaroglu”
Rcccivcd71 July 1991 Free rudi&induscd damegc to DNA in vivo is implicated to play II role in cwcinogcncris. Ewidcncc exists thal DNA dnmnpc by cndoynous free radiuls occurs in vivo, und thcrc is a rtcadpstatc lwcl of free radic&moditIcd hascs in ccllullrr DNA. WC hove invaligPtcd cndogcnous lcvcls of typical free rztdic&induscd DNA bits modifications in chromriz ofvarious human cancerous tissuesand their canswfrec rurrounding~issucr. Five different lypcr of sur@cally rcmovcd tirsucr were UK& nttmcly eolon, stomach. ovary, brain and lung tissues. In chromntin samples ivolatcd fram thcsc tissues. iivc pyrimidinedcrivcd and six purinc-dcrivcd modillcd DNA busts wcrc idcntincd and quantitatcd by ~schromatographyltnass spsctromctry with rslcctcd.ion monitoring, Thcsc wcrc 5.hydrox~S~mcthylhydantoin, 5.hydroxyhydantoin, 5~(hydroxymcthyl)urcil, S.hydroxycy. torinc, 5,6.dihydroxycyt6dac. 4.6~diamino.S-formnmidopyrimidinc. t.hydr.rxyudcninc, xnnthinc, I.hydronyodcninc, 2,6diaminc&hydron~5. furmnmidopyrimidinc, and 8*hydroxy8uaninc. That compounds arc known to bc formed typically by hydroxyl rsdisal attach on DNA bw. In all ems, clcvatcd umounts over control lcvcls of modifIcd DNA bares WCFC found in cancerous tirrucr. The umounta of modified beset dcpndcd nn the tiwc type, Lung tissues rcmovcd from mokcrs hlrd the highest incrcnrlr of modified basesubovcthe conrrol Icvcls,and the hifisst overall umounts, Colon cunscr tissue samples hurl the lowcrt lncrcuscs of mdiClcd bttscs over the control Icwls. The results clearly indicate hi&r rtcady&!tc lcvclr of moditlcd DNA basss in canceroustirsucr thaw in their cunccr.frcc surrounding tirruss~Some of thcsc l-ions arc known lo tx promutagenic. although others have not been invcrriylltcd for their mutpycnicity, Identified DNA lctions mny play B cwsativc role in atrcinoycncr!r. Oxidtrtivc damage; Free radicals; DNA damqc; Hydroxyl radial;
1. INTRODUCTION Oxygen-dcrivcd spcsics such its superoxidc radical (0,“). HJ&, singlet onygcn and hydroxyl rlrdical (‘OH) arc well-known to bc cytotoxic, and they may bc implicatcd in the etiology of a number of human discnacs including cancer (rcvicwcd in [I ,2]). The superoxidc radical is formed in almost all aerobic cells 131.Any tiring system producing Oz” is expected to produce HIOa by chemical or enzymatic dismutation of 07’-. Endogenously generated oxygen-derived species may cause damaBe to biological molcculcs, including DNA, by a variety of mechanisms (reviewed in [4]). Under physiological conditions, howcvsr, ncithcr 01” nor HiOI appears to produce modifications in DNA unless Curre~p~m/cnccullrlrcrr: M. Dizdaroglu, Chemical Licncc und Tcchnology Luborutory. National Inslilutc of Standards tind Tcchnolosy. Gaithcrsburg, MD 20899. USA, Fax: (I) (301) 330 3447, Oi-, supcroxidc radical; ‘OH, hydroxyl radical; 5.OH. SMc.Hyd. 5~hydrory~5~mcthylhydtintoin; S.OH.Hyd, S.hydronyhp dantoin; 5.01~~Cyt, 5~hydroxycytorinc; 5.OHMc.Ura, S(hydrox. ymcthyl)urdcil; 5.6.diOH-Uru. SP~dihydroxyurucil; FupyAdc, 4,6& amino-Sformamidopyrimidinc; &OH.Adc, t.hydroayadcninc; 20 P+W ‘+ydfO+~&&fIieSi F+l~#h& a44hmiae4hy~r~xy-5-fan . ..a ._. tf.OHXiua, mamidopyrimidinc; %hydroxyguaninc; USTFA, bir(trimcthylsilyl)trifluoroacctamidc: GUMS.SlM, gas chrometoga. phy/mass spcctromctry wilh sclcctcdaion monitoring. Abbrcviuthtr:
Mutation
metal ions arc present in the system [1,4]. Thus much of the toxicity of O,*- and H,Q is thought to result from their mcts\l ion-catalyzed conversion into highly reactive *OH [1.4], The hydroxyl radial produces o unique and extensive pnttcrn of chemical modifications in DNA and nuclcoprotcin, including modified bases and DNAprotein cross-links (rcvicwed in [4-71). Such DNA lcsions may be promutagcnic and may play a role in cnrcinogcncsis [ 1,4,8,9], Evidence exists that DNA damage by cndogcnous free radicals occurs and accumulates in viva, nnd that there is a stcady=statc level of free radical-modified base in cellular DNA [l&16]. Continuous cndobcnous demugc to cellular DNA by free radicals and accumulation of such damage has been suggcstcd to significantly contribute to carcinogen&s in humans [17-191. Bccausc of their ability to damage DNA, free radicals arc thought to be involved in all stogccsoictircinogencsis [2,4,9], Understanding the role of free radicals at the molecular lcvcl may lcod to an understanding of cancer related to free radicals. Such an understanding in turn will dspcnd on the charactcrization of the nature of DNA modifications caused by free radicals in living systems. Chemical characterization of free radical-induced . * iiI&tiit3iiS
ti ESiiIi&iZ
Zti
p&X
%SZ
ti
QNP,
can be achieved by a method incorporating the tcchnique of gas chromatography/mass spcctromctry with 193
Volume 309, number 1
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sclcctcd-ion monitoring (GC/MS-SLM), which cun bc applied to DNA it&f or directly to chromotin [7,20,2 11, By this method. u Iargc number of pyrimidinc- und purinc-derived modified buses has been shown to bc cndogcnously present in chromatin of cultured mummnliun cells, including human cells [22-251. The sumc mclhod hr~s recently been nppiicd to show incrcuscs over control lcvcls in the imlounts of four purinc-dcrived modified bslscs in DNA from ncoplitstic livers of feral fish cxposcd to czuxinogcns [X-28]. tend in the amounts of chrcc purinc-derived modified btrsco in DNA from cancerous fcmulc brCi\st tissues [29]. In the prcscnt work, WChove invcstifitcd cndogcnous lcvcls of cypicul free nrdicul-modified pyrimidincs und purines of DNA in chromsrtin sumplcs isolated from various human anccrous tissues and their cancer-free surrounding tissues. 2. MATERIALS AND METHODS 2.I, Ylrrrriulr
CCMiIl commercial cquipmcnl Or nralrrinh wc idcnliliid in this paper in order to specify adcquntcly rhc cxprimcntill procedure. Surh idcntifialtion dacs no1 imply rccomnundnlion or cndorwncm by the Nrlional lnslilulcofdt;lndardr llnd Technology. nor dacs it imply that the matcrisIs or c@pmcnl identified arc ncccwrily ~hc best nvailnblc Tsrthe purprrsc. Aulhenlic compounds wcrc purchossd or syntlrcsizcd as dcrrribcd previously [2?.23]. Xanthinc WIT purchased lrom Sigmu Chcmiwl Co. Acctanitrilc und bir(trimcthylsilyl)trifluorot~cctamidc (DSTFA) conruining 1% trimcthylcblarosihnc were abtaincd from Pierce Chcmicul CO. Formic ;dd WAS from Mnllinckrodt.
2.2. Nurrlull lismrs nnrl isolctriar~ uf riwaartrlir~ The human [issues wcrc obtain-d l?cn Dcprrfmcnls of Surgery. Mcdialt Schaal. Bydgasm. Dolmd. during surgery on cancer paticnls. Approximately l-l ,5 8 of tissues wcrc collcc~cd Tar ench chromalin isolation, AT&r surgikll rcmovul, WWWI riaucs and lhcir rcspscrivc surgicnl rnllrgins wcrc quickty ;IJ,.# ., in liquid nitrogen nnd maintained in thircondition until the isolation ofchromulin. Hirtopnrhologic& cvuluatian of surgically removed tissues rcvclrlcd thaw the surgiWl milrgin tisuer: wcrc tumor free, whilcwnccroux tissues cxhibilccl the l’ollawingcharactcrirticr: bruin cnnccr (millc p;iticnl). librillury aaracytoma; luny camccr 1 (l’cmidc patient. smoker), squumour cell wrciname; lung cancer II (Tcmtilc patient. smoker). adcnocarcinomx stomach anccr (male puticnt), mucinour wrcinomtl: ovary CBIICEF (f’cmirlc p;\ticnt). scrous cyrudcnacurcinomrl colon cancer (male pnticnt). lymphogrtinulomu (Hod&kin’s lymphnmn). Except for coltin ~xcr, nil Iuinors were primary tumors. Equul umounts (I g) 0r cunccrous tissues and thclr surrounding cunccr-l?cc tisrucs were uacd for chromulin isolation. Five scparnrc tirsuc samples collcclcd Tram the same lisruc lypc wcrc used. Frozen tissues were ground in porc&in grindcru, and then aurpcndcd in sucrasc buffer (0.25 M sucrose, 3 mM C&I,, 0.1 mM phcnylmcihuncrulku?yl fluoride, 0.1 mM dithie thrcitol, SO mM Tris-HCI UC pH 7.4) and homogcnizcd in il Tcflonglass homogcnizcr. Subrcqucnlly. samplcx wcrc lilkrcd through u nylon sicvc. Chromalin was then isolutcd us described previously [22]. 2.3. N_vWJTti. trLtrtl~.~isi~ylcttl~tt ttllci jpis cllrasl;tt~~ru~~fry/~tlcI~~ spccrrofNctr~ 7s a:kpts Gf diimtili XliTpks fin ! Inpi: fti lxiff~: zzn!t%z 0. I mg of DNA, I nmol of&azaihyminc and 2 nmol ol+S.ar*ludcninc wcrc added as inurnal slandardu, Snmplcs wcrc lhcn lyophilitcd. Samples ofchromutin wcrc hydrolyzed with 0.5 ml orGO% formic acid in cvacualcd and aculcd Iubcr ut 14O.C ror 30 min [15]. Samples wcrc
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Scptcmbw 1991
lyophilhcd nnd then trimc~hylsilyhtcd with (3. I ml of t( USTFA/xclonilrilc WI: v/v) mixlurc in polp(lr~rirfluoro~elhylcnc)~~p~d hypoviulr under nitrogen tit I 30X Tar 30 min. Anulpir of dcriwlixcd sumplcs wax prl’ormcd by GCXMS-SIM as dcrcribctl previously [X25], An aliquo~ (4 ~1) of cxh &xiv&cd sample was injcckd without nny further trcnlmcnt ima the injection pan of the gns chromnlogrnph by mcuns or;ln autosumplcr. A split rAtioof I:?0 wi~s used. rcvultiny in ti.2pyof hydrolyszd and dcrivrliued RNA #oiay through the GC column Tar each unuly%b.
3. RESULTS The objective of this work wus to cxtlminc whcthcr typical free rudicol-modified pyrimidincs nnd purincs occur in chromtrcin of vlrriour human cancerous tissues and their cunccr-free surrounding cissucs, und to compare the quunticics of these modified DNA bases in both types of tissues, Previously. clcvutcd nmowts over control lcvcls of three purine-derived modified bnsss in DNA isolated from cancerous tissues of human female brcilst were observed [19]. This study was limited to determination of three purinc lesions in DNA isolated from one cypc of human canrxrous tissue. DNA 8s n whole was isolated and analyzed. In the prcsfnt study, by contrast. WChave extmiinccl chromacin isolutcd from five different humon anccrous tissue i\nd [heir conccrfree surrounding tissues, Moreover, five pyrimidinc-dcrived and six purinc-derived modified buses wcrc idcntifitd and qutrncitatcd. The modified bnscs and chcir qunntitics arc given in Tublcs I and II, We also scnrchcd for two other products. namcty thyminc glycal and Shydroxyurucil. howcvcr. we were unable co identify the prcscncc of thcsc compounds in the tissue samples examincd. Fig. 1 illustrates the structures of the modified buses dealt with in the present work. 5Hydroxycytosinc (5.OH-Cyt) and T-hydroxyuracil result from ncid-induccd rnodificrrtion of cytosinc glycol: the former by dehydration and the latter by dcnminntion and dchydration [30]. Similxly. §,6-dihydroxyurucil (§.G-diOHUru) is formed by dcaminution of 5.6.dihydroxycytosine [30]. In addition to the modified bases found in chromutin in our previous publications [22-251, WChave identified xanthinc in the present work. This compound m;\y arise from dctiminution ofguonine, howcvcr, thcrc iu ulso a possibility that it may be formed by attack of ‘OH ut the carbon-2 of guaninc, Luq and colon cunccrous tissues had chc highest clnd the lowest increases. rcspectivcly. in the amounts of modified bases over the control amounts (Tables I und II). Cancer-free lung tissues from two patients had almost equal amounts of each modified base with rhc exception of two, namely 5-OH-S-Mc-Hyd and 5,6diOH-Cyt (Table I). Up to a7-fold increases in the amounts of both pyrimidinc- and purins-derived lesions __.___-__ wcrc obscrvcd in cancerous iung lissu& wifn rapcst to their cancer-free surrounding tissues. 111 terms of incrcascd amounts of lesions, there wcrc differcnccs bccwcen the two lung tissues examined. Essentially no
Volume 309. number 1
September 1992
FEBS LETTERS Tubls I Amounts’
5.0~~J-MC.Hyd S.GH-Hyd S-UHMcJJn 5.OH-Cyt 5.6-diQl-I- Ura FupyAdc txwAdc XInthine 2.OH-Adc FupyGua tl-OHGua
al’ modified DNA
barr (molcculcr per 10’ DNA
bass) in humnn Iissues
Control
Cvnccrous
Cmlrol
Cow01
I s65f0.09 1.77io.04 0,d I kO.07 l,OlfO.ld 0,49kO.O8 0.55*0.04 0.39co.05 5,%6?0.41 0,27to,a4 I ,03+0.07
2.015Mb d.3S+O.W 0.30,*0.06 1.16f0.23 0.99t0,17* 0.94+o.a4* 1.1710.25* 7.11010.73 l.lu~o,!L* 3,30+0,61* 2.dtkO.47'
1.82&O. I7 ?.L17+0.60 0.7dfO.clZI 0.67iO.13 0.3lfO.05 O,UiO.O6 3.06+O.IS 3.35kO.32 0.x*,0.21 1,17t0,1? 9,62ko.d6
6,10+0.55 3.75to.39 0.7l.tO.07 O.dO~O.07 l.lbtO.l8 0.74io.05 3,11+0,10 ul9t1.06 I.IJLO27 0.96tO.IO 7.33+0,97
I .mo.oa
‘bch vrluc rcprcscntx IIIC mean ? S.E.M. fram mcusurcmsnts ol’chromatin lSi~nillwntly difkrcnl from controls (P * 0.05 by Student’s I-tcs~).
insrcuscs wcrc obscrvcd in the amounts of 8-hydroxypurincs. ix, &OH-Adc and 8-OH-Guir. in lung cunccrous tissue 1. In both lung ctinccrous tissues, the highest incrcascs over the control levels were in the umounts of 3,-OH-Adc (a7- and a%fold). The lung conccrous tissue II hud the highest mountri of almost all modified bases when compared with othrr types of canccrs cxomincd, Stomach. ovary und brain auxcrous tissues tllso hud significantly hiyhcr amounts of lesions in their chrornutin than their rcspcctivc sonccr=frcc surrounding tissues. In thcss CUSSS,the highest incrcsrscs over control lcvcls wcrc observed in the amounts of &OH-GUI (&fold). S-OH-Cyt (s5-fold) and Z-OHAdc (a4-fold), rcspcctivcly. Colon cancerous tissue had significcrnt incrctiscs of modified bliscs only in 11few instnnccs. In most casts, the amount of cnch modified bnsc was similar in cancer-free tissues. The most varia-
sampler isoli~tcd I’rom five sqwatc
10.5~127' ld,5tl,39' 1.03*0.11* I .7i-lio.25* 3.t17*0.70* I .d7tO.?O' tMdf0,85' l5.3*2.61* 6.0Ifl.27* 3.0710.6tP 23.014.w
tirruc rampla.
blc amounts of modified buss in cancer-free tissues wcrc those of 8=OH-Adc and 8-OH=Guu, ranging from 0.39 to 3.11 S-UH=Adc/lO’ DNA bases, und from 0,94 to 9.68 8.OH-Gua/lO’ DNA bases. The amounts of 8-hydroxypurincs wcrc higher thun those of formtlmidopyrimidincs in both cancer-free and canscrou~ tissues of lung, stomuch. and awry. Colon and brain tissues had similar amounts of these compounds. Thcsc types of D??,‘?jxudiicts may urisc from rcspcctivc onc-clcctron oxidution tend reduction of carbon-&OH adduct rirdicals of purincs, which UC formed by the addition of ‘OH to the carbon-8 of purincs 1311. 4. DISCUSSION The results obt;rincd in the present work indiwtc that typical ‘OH-mediated products of DNA bases occur in
Tublc I I Amounts’
of modified DNA
buses (molcculcs per IO5 DNA slolllncla --
Colon lh5C
5.OH-~-MC-I-lyd 5.OH-Hyd 5.OHMcJJra 5.OH.Cyt 5.6.diO#-Uru FiipyA&
&GH-Adc Xunthinc 2.OH-hdc FapyGuu 8-OH-GUti
bass) in human tirrucr ovury
Control
Cilnccrour
Control
Canwous
Control
Cunccrous
2.67fo.22 I .69+0,23 0.39t0.01 0.44f0.03 0.4liO.09 O.t171O*IS 100620. I I 5.I7*0,8s 0.61f0.11 2.27kO.07 2.7120.13
4.lt1+0.27* 3,34+0.41 l 0,47+0.09 0.54+O.Ct6 0.9?+0.06’ 0.74*0.17 I ,34kO,?6 4.13*0.41 0.64a I I 2.66tO.72 4.43kO.37’
2.01~0.29 I .8710. I4 O,l3kO.O3 0.69+0.07 0.46+0.09 0.35+0.03 0.70*0. I2 217~0.46 C1.23kO.06 a.sS*o.uY 0.94fO. I9
3.19?ro.79 3.27f0.04* OXfO.Oi* 0.59to,O9 I ,29*0.32* 0.8 I *0.0?* 2.32kO.37* 5.3310.96* 0,4?kO,OP I ,65iQ.z= s,os* l&O?’
1.95+0.20 3.4Bf0.75 0.6320. I9 0.25~0.06 0,43f0,08 0.81+0*19 1.49f0.31. 6.14+0.§1 0.50+0.07 i .%F. i3 3. I I kO.71
6,42fO.78’ 12.7*2.36* 3.17PO.W I .35fO.W I .5SkO.44’ I,OtlfO.l1 3.32fO.W 9.68~1.13. I .66*0.07* ??%%W %?of?.43*
%uch vuluc rcprcrcntti the mcun f S,E.M, from mcusurcmcnis of chromstin ‘Siyliiiicanlly difkrcnt from conlrols P 4 0.05 by Studcnl’s r-test).
rillmplcs isalad
from five licparalc lirruc a;ln~plCS.
195
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FEES LE-ITERS
30% number ?
September 1991
544ydrory.5.rnMhyl. hydanwl
Fig. 1. SIructurcr of the modiflcd barer.
chromrrtin of both cancer-free and cancerous human tissuesof various types. In all six tissue types examined,
the cndogcnous amounts of most pyrimidine- and purinc-dsrived DNA lsaions in canssrous tissues were found to be consistsntly higher than in their rcspsctive cancsr-free surrounding tissues. Our results arc consis* tent with a rcccnc observation that the amounts of thrcs purinc-derived modified bases in cancerous tissues iso= lated from breasts of several human fsmalc patisnts were higher than control lsvcls [29], however, our study was not limited to one typs of tissus or to a limited nurnbcr of purinc-derived Mans. WC have cxamincd six types of tiesues, and have identified and quantitatcd eleven modified bases. In the present study, the increases in the amounts of modiFlcd basss over the control levels wcrc no more than M-fold in all tissue samplcs examined, as oppossd to El?-Fold observed prcviousiy in one typs of fern& breast cancer tissue [Z$y,in contrast to the prsvious study mcntioncd above. WC have also Found significantly increased amounts of FapyAds over control levels in four types of tissues, 196
It is not known whether the DNA base Mona idcntificd in the present work play n role in carcinogcncsis, or arc Formed in grcatcr amounts in canscrous tissues than in thsir cancer-frsc tissues as a result of the discusc. There is little doubt that free radicals and DNA damugc resulting from free radical reactions play an important role in carcinogcncsis (rcvierNcd in C1.321).In the same context, a number of free radical-induscd DNA base lesions have bcsn cxumincd for their biologisal conscqucnsce, and some of thsm have been Found to posa~ mutagenic propcrrics [8,33+. Ths majority of modified bases idcntifisd in the prcssnt work, however, have not been investigated for their biological conscqucnscs. IC is conceivable that these lesions, too, may be promutagcnic, DNA lesions measured hcrc (except For xanthins) arc known to bs produced in DNA (OSa whols) or in chromatin of mammalian cslls upon trcatmsnt of cclis with mutagenic agents such as hydrogen psroxidc or ionizing radiation [11,24,2S,41-48]. All these fasts mcntionsd above indicate a possible causative role for frcs radical-modified basss in carcinogcncsis.
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Eicvaicd icvcls of modified bases in canserous tissues may bc due to the production of Iarge amounts of H& which has been found to be characteristic of human tumor cells [49]. It is known that HzQ2 treatment of mammalian cclis causes formation of the herein idcntificd DNA lesions in their chromatin, most likely via sitc.spceifis ‘OH production [%?I. Furthermore. cvidcnce exists that tumor cells have abnormal lcvcis and activities of antioxidant cruymcs when compared with their rcspcctivc normal cells (reviewed in [32]!. Low lcvcls of antioxidant enzymes, such aa supsroxidc dismutasc or cataiase. in tumor c&s mny cause accumulation Of Oi- and Hz% with subsequent ‘OH-induced damage to DNA, resulting in grcatcr amounts of mod= ified DNA buses in tumor cells than in normal ecils. Diffcrcnccs in the activities of antioxidant cnzymcs among individual tumors may account for the different amounts ot’ modified DNA bases that were found in various cancerous tissues in the present work. In this rcspcct, two lung cancerous tissues hnd the greatest amounts of modiflcd DNA bases among the tissues cxamincd hcrc This may be attributed in part to the fact that both patients were heavy smokers. Epidcmioiogicai studies have shown that smoking is the major cause of human lung eanecr [SO].Cigarette smoke is known to cause DNA damage in human lung ceils and cigarette smoke-induced generation of H202 with subscqucnt formation OF‘OH has been implicated in causing DNA damage [Sl-S3j. The types of role that individual free radical-induced DNA base modifications play in carcinogcncsis arc not known, and remain to bc dctcrmincd. High lcvcls of modified DNA bases observed in various human tumor cciis may be in part a result of the disease. Whatever the reason is for this phcnomcnon, high lcvcis of modified DNA bases may contribute to the gcnctic instabiliiy of tumor cells, and thus to increased mctastatic potential [54,55]. Epidemiological studies involving measurement of typical free radical-modified DNA bases in a large variety of individual tumor tissues zu~dtheir respective normal tissues may provide insight into mechanisms of carcinogcnesis related to oxygen-derived spccics. Mens= urcmcnt of pyrimidinc- and purine-derived DNA Icsions in tissues as described herein may prove to be useful in determining an association bctwccn free radical-producing agents and cancer risk. Ae~knon~l~~~~trc~as: M.D. gntcfulty
acknowledges partial finnncinl support from the Office of Clculth and Environmental Rcscarch, Oflice of Energy Rcscurch. US Dcpartmrnt of Energy, Washington, DC.
REFERENCES f4] Halli~& 8. an8 GuW&&, d,Fl.C.(1989)Fr@ Rdi& Biology and Mcdicinc, 2nd cdn,, Clnrcndon Press, Oxford. [a] Ccrutli, P/r, (1985) Scicncc 227, 375-381 I [3] Fridovich, I. (1986) Arch. Biochcm. Biophys. 247, I-l I. [4] Helliwcll, 6. and Aruoma, 0.1. (1991) FEDS Lctt. 281, 9-19,
in
September
1992
[5] Von Sonntog, C. (1987) The Chsmicorl Busis of Radiation Biolm opy. pp, 116-166 and 221-294, Taylor & Francis, London. [6] Olcinick, N-L., Chiu, S., Ramukrishnnn. N. and Xuc, L (1987) Br. J. Cunfcr55 (ruppl. VIII), 135-140. [7] Diadirroylu. M. (1991) Free Rud, Biol. Med. 10,?25-242. [II] Brcimcr, L,H. (1990) Mol. Carcinagcnais 3, 168-197. [O] Floyd, R.A. (1990) FASEEJ J. 4. 2587-2597. [IO] Cuthcart, R., Schwicrs. E., Snul, R.L. and Amcr, B.N. (1984) Prac. NntL Acud. Sci, USA 81. 5633-5637. [i I] Kusai, H.. Grain, P,F., Kushino,Y,, Nisbimun, S., Ootsuyama, A. und Tanooko, H. (1986) Cnrcinogrnair 7, 1849-1851. [I21 Adclnum. R,, Saul, R.L. and Amcr, B.N. (1988)Droc. Natl. Acad, ki. USA 85, 27062708. (131 Richter, C.. Park, J.-W. and Ames, B.N. (1988) Pros, Nell. Acad, Ssi. USA B5,6465-6467. Slillwcll, W.G., Xu. H.-X., Adkins, J.A.. Wishnok, J.S. andTanncnbuum, S.R. (1989) Chcm. Rcs. Tonicol. 2.94-99. Fruga. C.G., Shincgu, M-K., Park, J.-W., De&an, P. nnd Ama, B.N. (1990) Proc. Nell. Aud, Oi, USA 87,453345$7. Fmgu. C.G., Motchnik. D.A.. Shinrg;r, M.K.. Hclbock. H.J., Jacob. R.A. and Amer. B.N. (1991) Proc, Natl. Acad. Ssi. USA 88, 11003-l 1006. Totter. J.R. (1980) Proc. Natl. Acad. Sci, USA 77. 1763-1767. Amer. B.N. (1983) Scicncc 221, 1256-l 264. Ama, U.N. (1989) Free Rnd. Rcs. Commun. 7, 121-128. Dizddnroglu, M. (1985) Anal. Biochcm. 144, 593-603. Dizdaroglu. M. (1990) Methods Enzymol. 193, 842-857. Gajcwrki, E.,,Rae, G,, Nackcrdicn, 2. and Dixdrro,@u. M. (19YQ Biochcmirtcy 29. 78767882. Nsckcrdirn, 2.. Kasprpnk. K.S., Ruo, G.. Hnlliwcll, B. and Dizdnroglu. M. (1991) Canax Rcs. 51, 5837-5842. Dizdaroglu, M., Nackcrdicn, 2, Chao, B.C., Oajewrki, E. and RPO,G, (1991) Arch, Biuchcm. Biophys. 285, 3X8-3PO. Nnckcrdicn, 2.. Olinrki. R. rind Dizdrroglu, M. (1992) Free Rud. Ra Commun. 16. 259-273. Mali& DC. und Hoimunot. R. (l99U) Biochcm. Biophyr, Rcr. Commun. 173.61~19. Malinr, DC., Ostrandcr, G.K.. Haimanot. R. rend Williams. P. (1990) Curclnogcntiis 11, 1045-1047. Mali& DC. und Haimanot, R. (1691)AqueticToxicol. 20. l23130. Mnlinr, D.C. and Haimunot, R. (1991) Cancer Rcu. 51. 54305432. Ditiuroglu, M., Holwitt. E.. Hagirn, H.P. tmd Blakcly. W.F. (1986) Diochcm. J. 235. 531-536. Stccnkcn, S. (1989) Chcm, Rev. 89, 503-520, [32] run, S, (1990) Free Rud. Biol. Med. 8, 583-599. [33] Wallncc. S.S. (1987) Br. 1. Cancer 55 (suppl. VIII). 118-125. [34] Shinumd.Mod. L., Roraman, T.G., Troll, W., Tccbur, G,W. nnd Frcnkcl, K. (1917) Mutat, Rcs. 178, 177-116. 1351Hnycs, RX., Pctrullo, L,A., HutinS, H., Wallxc. S.S. and Le. Clcrc, J.E. (1988) J. Mol. Biol. 201. 234-246. [36] Buau. A.K.. Lorchlcr, E.L., Leadon, S.A. and EsQmann. 1-M. (19119)Prac. NatI. Acad. Sci. USA 86. 7677-7681. [37] Wood, M-L.. Dizdaroglu, M,, Gajcwski, E. and Essigmann, J.M. 11990) Biochemistry 29, 7024-7032. [38] Moriyti. M.. Ou. C.. Bodcpudi. V., Johnson. F.. Takcshita. M. und Grollman. A.P. (1991) Mutat. R&x 254.281-288. [3Y] Chcng, KC., Cahill, D.S.. Kasai, H., Nishimura, S. and Losb. L-A, (1992) 1. Biol. Chcm. 267, 166-173. [40] Guschlbaucr, W., Da&a, A.-M.. Guy, A., Tcoulc, R. and Fazakcrlcy, G.V. (1Y91) Nucleic Acids Rce. 19, 1753-1758. [41] Harihutxm. P.V. and Ccrutti. P.A, (1972) J. Mel, Biol, 66. GZ-81, [42] Mattcrn. M.R., Harihann, P.V. und Ccrutti, P.A, (1975) Bib chim. Biophys. Astu 395.48-55. [43] Lmdon, %A, and Hanawalt, P.C. (1983) Mutat. Rcs. 112, IsI-, x?o. [44] Tccbor. G,W.. Frcnkcl, K, and Goldstein. MS. (1984) Proc, Nutl. Aad, Sci. USA 81, 318-321,
Volume 309. number 2
FEDS LETTERS
(451 Frmkcl. K.. Cumminp, A.. Solomon, J.. Chrlc~,J., Shhxy, J, and Tccbar. GW. (1985) Uiochcmistry ?4,45?74522. (461 R&w. L.H. and Lindnhl. T. (19115)Uiaehnblry 24. 40lf4022. [471 ptltil. M.S.. Larhcr. S.D. and Hurihurun. P.V. (1911%Int. J, Ra diut. WI. 48.691-700. [48) Ladon. S,A, (1990) Hculth Phyr. 59, 15-22. (491 Sxittroit3ki. T.P. ;lad Nathan. CF. (1991) Cancer Ra. 51. 791798. (50) US Dcparmcn~ nf Hcal~b Education und Wclfarc. Smokiny und Hculth, A Report of the Surgeon Gcncr;tl (IW) RHEW Publication, No, (PHS) 79.SOW, p. 3. 1
.
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WI P1dwww ‘I’., Kwke, M.. Ksdumu. M. und Nugu~u.C. (lN5) NwJrc 3 14, 462a4. I521 Prsar. W-A. (19871 Ur. f. Cunccr 55 (arppl. VIII), 1%.23. IS31 Lcrukkrsw. POnndTugwon.C, (1992)Chctwl3iol. 11%81. l97.208. 1541Cifonc. M.A. und Fidlcr. I.J. (1961) Proe. Nutl. Awd. Sci. USA 78.6949-4952. IS51 Mcyidish, T. und Mazurck. N.A. (1989) Netwe 342, 807-till.
Scptcmhr 1992
DNA base modifications in ckromatin of human cancerous tissues Ryszard OlinskPb, ‘f’omasz Zastawnyb, Jsroslaw Budzbon”, Jan Skokowskib,Wojtck Zqarskib and Miral Dizdaroglu”
Rcccivcd71 July 1991 Free rudi&induscd damegc to DNA in vivo is implicated to play II role in cwcinogcncris. Ewidcncc exists thal DNA dnmnpc by cndoynous free radiuls occurs in vivo, und thcrc is a rtcadpstatc lwcl of free radic&moditIcd hascs in ccllullrr DNA. WC hove invaligPtcd cndogcnous lcvcls of typical free rztdic&induscd DNA bits modifications in chromriz ofvarious human cancerous tissuesand their canswfrec rurrounding~issucr. Five different lypcr of sur@cally rcmovcd tirsucr were UK& nttmcly eolon, stomach. ovary, brain and lung tissues. In chromntin samples ivolatcd fram thcsc tissues. iivc pyrimidinedcrivcd and six purinc-dcrivcd modillcd DNA busts wcrc idcntincd and quantitatcd by ~schromatographyltnass spsctromctry with rslcctcd.ion monitoring, Thcsc wcrc 5.hydrox~S~mcthylhydantoin, 5.hydroxyhydantoin, 5~(hydroxymcthyl)urcil, S.hydroxycy. torinc, 5,6.dihydroxycyt6dac. 4.6~diamino.S-formnmidopyrimidinc. t.hydr.rxyudcninc, xnnthinc, I.hydronyodcninc, 2,6diaminc&hydron~5. furmnmidopyrimidinc, and 8*hydroxy8uaninc. That compounds arc known to bc formed typically by hydroxyl rsdisal attach on DNA bw. In all ems, clcvatcd umounts over control lcvcls of modifIcd DNA bares WCFC found in cancerous tirrucr. The umounta of modified beset dcpndcd nn the tiwc type, Lung tissues rcmovcd from mokcrs hlrd the highest incrcnrlr of modified basesubovcthe conrrol Icvcls,and the hifisst overall umounts, Colon cunscr tissue samples hurl the lowcrt lncrcuscs of mdiClcd bttscs over the control Icwls. The results clearly indicate hi&r rtcady&!tc lcvclr of moditlcd DNA basss in canceroustirsucr thaw in their cunccr.frcc surrounding tirruss~Some of thcsc l-ions arc known lo tx promutagenic. although others have not been invcrriylltcd for their mutpycnicity, Identified DNA lctions mny play B cwsativc role in atrcinoycncr!r. Oxidtrtivc damage; Free radicals; DNA damqc; Hydroxyl radial;
1. INTRODUCTION Oxygen-dcrivcd spcsics such its superoxidc radical (0,“). HJ&, singlet onygcn and hydroxyl rlrdical (‘OH) arc well-known to bc cytotoxic, and they may bc implicatcd in the etiology of a number of human discnacs including cancer (rcvicwcd in [I ,2]). The superoxidc radical is formed in almost all aerobic cells 131.Any tiring system producing Oz” is expected to produce HIOa by chemical or enzymatic dismutation of 07’-. Endogenously generated oxygen-derived species may cause damaBe to biological molcculcs, including DNA, by a variety of mechanisms (reviewed in [4]). Under physiological conditions, howcvsr, ncithcr 01” nor HiOI appears to produce modifications in DNA unless Curre~p~m/cnccullrlrcrr: M. Dizdaroglu, Chemical Licncc und Tcchnology Luborutory. National Inslilutc of Standards tind Tcchnolosy. Gaithcrsburg, MD 20899. USA, Fax: (I) (301) 330 3447, Oi-, supcroxidc radical; ‘OH, hydroxyl radical; 5.OH. SMc.Hyd. 5~hydrory~5~mcthylhydtintoin; S.OH.Hyd, S.hydronyhp dantoin; 5.01~~Cyt, 5~hydroxycytorinc; 5.OHMc.Ura, S(hydrox. ymcthyl)urdcil; 5.6.diOH-Uru. SP~dihydroxyurucil; FupyAdc, 4,6& amino-Sformamidopyrimidinc; &OH.Adc, t.hydroayadcninc; 20 P+W ‘+ydfO+~&&fIieSi F+l~#h& a44hmiae4hy~r~xy-5-fan . ..a ._. tf.OHXiua, mamidopyrimidinc; %hydroxyguaninc; USTFA, bir(trimcthylsilyl)trifluoroacctamidc: GUMS.SlM, gas chrometoga. phy/mass spcctromctry wilh sclcctcdaion monitoring. Abbrcviuthtr:
Mutation
metal ions arc present in the system [1,4]. Thus much of the toxicity of O,*- and H,Q is thought to result from their mcts\l ion-catalyzed conversion into highly reactive *OH [1.4], The hydroxyl radial produces o unique and extensive pnttcrn of chemical modifications in DNA and nuclcoprotcin, including modified bases and DNAprotein cross-links (rcvicwed in [4-71). Such DNA lcsions may be promutagcnic and may play a role in cnrcinogcncsis [ 1,4,8,9], Evidence exists that DNA damage by cndogcnous free radicals occurs and accumulates in viva, nnd that there is a stcady=statc level of free radical-modified base in cellular DNA [l&16]. Continuous cndobcnous demugc to cellular DNA by free radicals and accumulation of such damage has been suggcstcd to significantly contribute to carcinogen&s in humans [17-191. Bccausc of their ability to damage DNA, free radicals arc thought to be involved in all stogccsoictircinogencsis [2,4,9], Understanding the role of free radicals at the molecular lcvcl may lcod to an understanding of cancer related to free radicals. Such an understanding in turn will dspcnd on the charactcrization of the nature of DNA modifications caused by free radicals in living systems. Chemical characterization of free radical-induced . * iiI&tiit3iiS
ti ESiiIi&iZ
Zti
p&X
%SZ
ti
QNP,
can be achieved by a method incorporating the tcchnique of gas chromatography/mass spcctromctry with 193
Volume 309, number 1
FERS LEl-TERS
sclcctcd-ion monitoring (GC/MS-SLM), which cun bc applied to DNA it&f or directly to chromotin [7,20,2 11, By this method. u Iargc number of pyrimidinc- und purinc-derived modified buses has been shown to bc cndogcnously present in chromatin of cultured mummnliun cells, including human cells [22-251. The sumc mclhod hr~s recently been nppiicd to show incrcuscs over control lcvcls in the imlounts of four purinc-dcrived modified bslscs in DNA from ncoplitstic livers of feral fish cxposcd to czuxinogcns [X-28]. tend in the amounts of chrcc purinc-derived modified btrsco in DNA from cancerous fcmulc brCi\st tissues [29]. In the prcscnt work, WChove invcstifitcd cndogcnous lcvcls of cypicul free nrdicul-modified pyrimidincs und purines of DNA in chromsrtin sumplcs isolated from various human anccrous tissues and their cancer-free surrounding tissues. 2. MATERIALS AND METHODS 2.I, Ylrrrriulr
CCMiIl commercial cquipmcnl Or nralrrinh wc idcnliliid in this paper in order to specify adcquntcly rhc cxprimcntill procedure. Surh idcntifialtion dacs no1 imply rccomnundnlion or cndorwncm by the Nrlional lnslilulcofdt;lndardr llnd Technology. nor dacs it imply that the matcrisIs or c@pmcnl identified arc ncccwrily ~hc best nvailnblc Tsrthe purprrsc. Aulhenlic compounds wcrc purchossd or syntlrcsizcd as dcrrribcd previously [2?.23]. Xanthinc WIT purchased lrom Sigmu Chcmiwl Co. Acctanitrilc und bir(trimcthylsilyl)trifluorot~cctamidc (DSTFA) conruining 1% trimcthylcblarosihnc were abtaincd from Pierce Chcmicul CO. Formic ;dd WAS from Mnllinckrodt.
2.2. Nurrlull lismrs nnrl isolctriar~ uf riwaartrlir~ The human [issues wcrc obtain-d l?cn Dcprrfmcnls of Surgery. Mcdialt Schaal. Bydgasm. Dolmd. during surgery on cancer paticnls. Approximately l-l ,5 8 of tissues wcrc collcc~cd Tar ench chromalin isolation, AT&r surgikll rcmovul, WWWI riaucs and lhcir rcspscrivc surgicnl rnllrgins wcrc quickty ;IJ,.# ., in liquid nitrogen nnd maintained in thircondition until the isolation ofchromulin. Hirtopnrhologic& cvuluatian of surgically removed tissues rcvclrlcd thaw the surgiWl milrgin tisuer: wcrc tumor free, whilcwnccroux tissues cxhibilccl the l’ollawingcharactcrirticr: bruin cnnccr (millc p;iticnl). librillury aaracytoma; luny camccr 1 (l’cmidc patient. smoker), squumour cell wrciname; lung cancer II (Tcmtilc patient. smoker). adcnocarcinomx stomach anccr (male puticnt), mucinour wrcinomtl: ovary CBIICEF (f’cmirlc p;\ticnt). scrous cyrudcnacurcinomrl colon cancer (male pnticnt). lymphogrtinulomu (Hod&kin’s lymphnmn). Except for coltin ~xcr, nil Iuinors were primary tumors. Equul umounts (I g) 0r cunccrous tissues and thclr surrounding cunccr-l?cc tisrucs were uacd for chromulin isolation. Five scparnrc tirsuc samples collcclcd Tram the same lisruc lypc wcrc used. Frozen tissues were ground in porc&in grindcru, and then aurpcndcd in sucrasc buffer (0.25 M sucrose, 3 mM C&I,, 0.1 mM phcnylmcihuncrulku?yl fluoride, 0.1 mM dithie thrcitol, SO mM Tris-HCI UC pH 7.4) and homogcnizcd in il Tcflonglass homogcnizcr. Subrcqucnlly. samplcx wcrc lilkrcd through u nylon sicvc. Chromalin was then isolutcd us described previously [22]. 2.3. N_vWJTti. trLtrtl~.~isi~ylcttl~tt ttllci jpis cllrasl;tt~~ru~~fry/~tlcI~~ spccrrofNctr~ 7s a:kpts Gf diimtili XliTpks fin ! Inpi: fti lxiff~: zzn!t%z 0. I mg of DNA, I nmol of&azaihyminc and 2 nmol ol+S.ar*ludcninc wcrc added as inurnal slandardu, Snmplcs wcrc lhcn lyophilitcd. Samples ofchromutin wcrc hydrolyzed with 0.5 ml orGO% formic acid in cvacualcd and aculcd Iubcr ut 14O.C ror 30 min [15]. Samples wcrc
194
Scptcmbw 1991
lyophilhcd nnd then trimc~hylsilyhtcd with (3. I ml of t( USTFA/xclonilrilc WI: v/v) mixlurc in polp(lr~rirfluoro~elhylcnc)~~p~d hypoviulr under nitrogen tit I 30X Tar 30 min. Anulpir of dcriwlixcd sumplcs wax prl’ormcd by GCXMS-SIM as dcrcribctl previously [X25], An aliquo~ (4 ~1) of cxh &xiv&cd sample was injcckd without nny further trcnlmcnt ima the injection pan of the gns chromnlogrnph by mcuns or;ln autosumplcr. A split rAtioof I:?0 wi~s used. rcvultiny in ti.2pyof hydrolyszd and dcrivrliued RNA #oiay through the GC column Tar each unuly%b.
3. RESULTS The objective of this work wus to cxtlminc whcthcr typical free rudicol-modified pyrimidincs nnd purincs occur in chromtrcin of vlrriour human cancerous tissues and their cunccr-free surrounding cissucs, und to compare the quunticics of these modified DNA bases in both types of tissues, Previously. clcvutcd nmowts over control lcvcls of three purine-derived modified bnsss in DNA isolated from cancerous tissues of human female brcilst were observed [19]. This study was limited to determination of three purinc lesions in DNA isolated from one cypc of human canrxrous tissue. DNA 8s n whole was isolated and analyzed. In the prcsfnt study, by contrast. WChave extmiinccl chromacin isolutcd from five different humon anccrous tissue i\nd [heir conccrfree surrounding tissues, Moreover, five pyrimidinc-dcrived and six purinc-derived modified buses wcrc idcntifitd and qutrncitatcd. The modified bnscs and chcir qunntitics arc given in Tublcs I and II, We also scnrchcd for two other products. namcty thyminc glycal and Shydroxyurucil. howcvcr. we were unable co identify the prcscncc of thcsc compounds in the tissue samples examincd. Fig. 1 illustrates the structures of the modified buses dealt with in the present work. 5Hydroxycytosinc (5.OH-Cyt) and T-hydroxyuracil result from ncid-induccd rnodificrrtion of cytosinc glycol: the former by dehydration and the latter by dcnminntion and dchydration [30]. Similxly. §,6-dihydroxyurucil (§.G-diOHUru) is formed by dcaminution of 5.6.dihydroxycytosine [30]. In addition to the modified bases found in chromutin in our previous publications [22-251, WChave identified xanthinc in the present work. This compound m;\y arise from dctiminution ofguonine, howcvcr, thcrc iu ulso a possibility that it may be formed by attack of ‘OH ut the carbon-2 of guaninc, Luq and colon cunccrous tissues had chc highest clnd the lowest increases. rcspectivcly. in the amounts of modified bases over the control amounts (Tables I und II). Cancer-free lung tissues from two patients had almost equal amounts of each modified base with rhc exception of two, namely 5-OH-S-Mc-Hyd and 5,6diOH-Cyt (Table I). Up to a7-fold increases in the amounts of both pyrimidinc- and purins-derived lesions __.___-__ wcrc obscrvcd in cancerous iung lissu& wifn rapcst to their cancer-free surrounding tissues. 111 terms of incrcascd amounts of lesions, there wcrc differcnccs bccwcen the two lung tissues examined. Essentially no
Volume 309. number 1
September 1992
FEBS LETTERS Tubls I Amounts’
5.0~~J-MC.Hyd S.GH-Hyd S-UHMcJJn 5.OH-Cyt 5.6-diQl-I- Ura FupyAdc txwAdc XInthine 2.OH-Adc FupyGua tl-OHGua
al’ modified DNA
barr (molcculcr per 10’ DNA
bass) in humnn Iissues
Control
Cvnccrous
Cmlrol
Cow01
I s65f0.09 1.77io.04 0,d I kO.07 l,OlfO.ld 0,49kO.O8 0.55*0.04 0.39co.05 5,%6?0.41 0,27to,a4 I ,03+0.07
2.015Mb d.3S+O.W 0.30,*0.06 1.16f0.23 0.99t0,17* 0.94+o.a4* 1.1710.25* 7.11010.73 l.lu~o,!L* 3,30+0,61* 2.dtkO.47'
1.82&O. I7 ?.L17+0.60 0.7dfO.clZI 0.67iO.13 0.3lfO.05 O,UiO.O6 3.06+O.IS 3.35kO.32 0.x*,0.21 1,17t0,1? 9,62ko.d6
6,10+0.55 3.75to.39 0.7l.tO.07 O.dO~O.07 l.lbtO.l8 0.74io.05 3,11+0,10 ul9t1.06 I.IJLO27 0.96tO.IO 7.33+0,97
I .mo.oa
‘bch vrluc rcprcscntx IIIC mean ? S.E.M. fram mcusurcmsnts ol’chromatin lSi~nillwntly difkrcnl from controls (P * 0.05 by Student’s I-tcs~).
insrcuscs wcrc obscrvcd in the amounts of 8-hydroxypurincs. ix, &OH-Adc and 8-OH-Guir. in lung cunccrous tissue 1. In both lung ctinccrous tissues, the highest incrcascs over the control levels were in the umounts of 3,-OH-Adc (a7- and a%fold). The lung conccrous tissue II hud the highest mountri of almost all modified bases when compared with othrr types of canccrs cxomincd, Stomach. ovary und brain auxcrous tissues tllso hud significantly hiyhcr amounts of lesions in their chrornutin than their rcspcctivc sonccr=frcc surrounding tissues. In thcss CUSSS,the highest incrcsrscs over control lcvcls wcrc observed in the amounts of &OH-GUI (&fold). S-OH-Cyt (s5-fold) and Z-OHAdc (a4-fold), rcspcctivcly. Colon cancerous tissue had significcrnt incrctiscs of modified bliscs only in 11few instnnccs. In most casts, the amount of cnch modified bnsc was similar in cancer-free tissues. The most varia-
sampler isoli~tcd I’rom five sqwatc
10.5~127' ld,5tl,39' 1.03*0.11* I .7i-lio.25* 3.t17*0.70* I .d7tO.?O' tMdf0,85' l5.3*2.61* 6.0Ifl.27* 3.0710.6tP 23.014.w
tirruc rampla.
blc amounts of modified buss in cancer-free tissues wcrc those of 8=OH-Adc and 8-OH=Guu, ranging from 0.39 to 3.11 S-UH=Adc/lO’ DNA bases, und from 0,94 to 9.68 8.OH-Gua/lO’ DNA bases. The amounts of 8-hydroxypurincs wcrc higher thun those of formtlmidopyrimidincs in both cancer-free and canscrou~ tissues of lung, stomuch. and awry. Colon and brain tissues had similar amounts of these compounds. Thcsc types of D??,‘?jxudiicts may urisc from rcspcctivc onc-clcctron oxidution tend reduction of carbon-&OH adduct rirdicals of purincs, which UC formed by the addition of ‘OH to the carbon-8 of purincs 1311. 4. DISCUSSION The results obt;rincd in the present work indiwtc that typical ‘OH-mediated products of DNA bases occur in
Tublc I I Amounts’
of modified DNA
buses (molcculcs per IO5 DNA slolllncla --
Colon lh5C
5.OH-~-MC-I-lyd 5.OH-Hyd 5.OHMcJJra 5.OH.Cyt 5.6.diO#-Uru FiipyA&
&GH-Adc Xunthinc 2.OH-hdc FapyGuu 8-OH-GUti
bass) in human tirrucr ovury
Control
Cilnccrour
Control
Canwous
Control
Cunccrous
2.67fo.22 I .69+0,23 0.39t0.01 0.44f0.03 0.4liO.09 O.t171O*IS 100620. I I 5.I7*0,8s 0.61f0.11 2.27kO.07 2.7120.13
4.lt1+0.27* 3,34+0.41 l 0,47+0.09 0.54+O.Ct6 0.9?+0.06’ 0.74*0.17 I ,34kO,?6 4.13*0.41 0.64a I I 2.66tO.72 4.43kO.37’
2.01~0.29 I .8710. I4 O,l3kO.O3 0.69+0.07 0.46+0.09 0.35+0.03 0.70*0. I2 217~0.46 C1.23kO.06 a.sS*o.uY 0.94fO. I9
3.19?ro.79 3.27f0.04* OXfO.Oi* 0.59to,O9 I ,29*0.32* 0.8 I *0.0?* 2.32kO.37* 5.3310.96* 0,4?kO,OP I ,65iQ.z= s,os* l&O?’
1.95+0.20 3.4Bf0.75 0.6320. I9 0.25~0.06 0,43f0,08 0.81+0*19 1.49f0.31. 6.14+0.§1 0.50+0.07 i .%F. i3 3. I I kO.71
6,42fO.78’ 12.7*2.36* 3.17PO.W I .35fO.W I .5SkO.44’ I,OtlfO.l1 3.32fO.W 9.68~1.13. I .66*0.07* ??%%W %?of?.43*
%uch vuluc rcprcrcntti the mcun f S,E.M, from mcusurcmcnis of chromstin ‘Siyliiiicanlly difkrcnt from conlrols P 4 0.05 by Studcnl’s r-test).
rillmplcs isalad
from five licparalc lirruc a;ln~plCS.
195
Volume
FEES LE-ITERS
30% number ?
September 1991
544ydrory.5.rnMhyl. hydanwl
Fig. 1. SIructurcr of the modiflcd barer.
chromrrtin of both cancer-free and cancerous human tissuesof various types. In all six tissue types examined,
the cndogcnous amounts of most pyrimidine- and purinc-dsrived DNA lsaions in canssrous tissues were found to be consistsntly higher than in their rcspsctive cancsr-free surrounding tissues. Our results arc consis* tent with a rcccnc observation that the amounts of thrcs purinc-derived modified bases in cancerous tissues iso= lated from breasts of several human fsmalc patisnts were higher than control lsvcls [29], however, our study was not limited to one typs of tissus or to a limited nurnbcr of purinc-derived Mans. WC have cxamincd six types of tiesues, and have identified and quantitatcd eleven modified bases. In the present study, the increases in the amounts of modiFlcd basss over the control levels wcrc no more than M-fold in all tissue samplcs examined, as oppossd to El?-Fold observed prcviousiy in one typs of fern& breast cancer tissue [Z$y,in contrast to the prsvious study mcntioncd above. WC have also Found significantly increased amounts of FapyAds over control levels in four types of tissues, 196
It is not known whether the DNA base Mona idcntificd in the present work play n role in carcinogcncsis, or arc Formed in grcatcr amounts in canscrous tissues than in thsir cancer-frsc tissues as a result of the discusc. There is little doubt that free radicals and DNA damugc resulting from free radical reactions play an important role in carcinogcncsis (rcvierNcd in C1.321).In the same context, a number of free radical-induscd DNA base lesions have bcsn cxumincd for their biologisal conscqucnsce, and some of thsm have been Found to posa~ mutagenic propcrrics [8,33+. Ths majority of modified bases idcntifisd in the prcssnt work, however, have not been investigated for their biological conscqucnscs. IC is conceivable that these lesions, too, may be promutagcnic, DNA lesions measured hcrc (except For xanthins) arc known to bs produced in DNA (OSa whols) or in chromatin of mammalian cslls upon trcatmsnt of cclis with mutagenic agents such as hydrogen psroxidc or ionizing radiation [11,24,2S,41-48]. All these fasts mcntionsd above indicate a possible causative role for frcs radical-modified basss in carcinogcncsis.
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Eicvaicd icvcls of modified bases in canserous tissues may bc due to the production of Iarge amounts of H& which has been found to be characteristic of human tumor cells [49]. It is known that HzQ2 treatment of mammalian cclis causes formation of the herein idcntificd DNA lesions in their chromatin, most likely via sitc.spceifis ‘OH production [%?I. Furthermore. cvidcnce exists that tumor cells have abnormal lcvcis and activities of antioxidant cruymcs when compared with their rcspcctivc normal cells (reviewed in [32]!. Low lcvcls of antioxidant enzymes, such aa supsroxidc dismutasc or cataiase. in tumor c&s mny cause accumulation Of Oi- and Hz% with subsequent ‘OH-induced damage to DNA, resulting in grcatcr amounts of mod= ified DNA buses in tumor cells than in normal ecils. Diffcrcnccs in the activities of antioxidant cnzymcs among individual tumors may account for the different amounts ot’ modified DNA bases that were found in various cancerous tissues in the present work. In this rcspcct, two lung cancerous tissues hnd the greatest amounts of modiflcd DNA bases among the tissues cxamincd hcrc This may be attributed in part to the fact that both patients were heavy smokers. Epidcmioiogicai studies have shown that smoking is the major cause of human lung eanecr [SO].Cigarette smoke is known to cause DNA damage in human lung ceils and cigarette smoke-induced generation of H202 with subscqucnt formation OF‘OH has been implicated in causing DNA damage [Sl-S3j. The types of role that individual free radical-induced DNA base modifications play in carcinogcncsis arc not known, and remain to bc dctcrmincd. High lcvcls of modified DNA bases observed in various human tumor cciis may be in part a result of the disease. Whatever the reason is for this phcnomcnon, high lcvcis of modified DNA bases may contribute to the gcnctic instabiliiy of tumor cells, and thus to increased mctastatic potential [54,55]. Epidemiological studies involving measurement of typical free radical-modified DNA bases in a large variety of individual tumor tissues zu~dtheir respective normal tissues may provide insight into mechanisms of carcinogcnesis related to oxygen-derived spccics. Mens= urcmcnt of pyrimidinc- and purine-derived DNA Icsions in tissues as described herein may prove to be useful in determining an association bctwccn free radical-producing agents and cancer risk. Ae~knon~l~~~~trc~as: M.D. gntcfulty
acknowledges partial finnncinl support from the Office of Clculth and Environmental Rcscarch, Oflice of Energy Rcscurch. US Dcpartmrnt of Energy, Washington, DC.
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(451 Frmkcl. K.. Cumminp, A.. Solomon, J.. Chrlc~,J., Shhxy, J, and Tccbar. GW. (1985) Uiochcmistry ?4,45?74522. (461 R&w. L.H. and Lindnhl. T. (19115)Uiaehnblry 24. 40lf4022. [471 ptltil. M.S.. Larhcr. S.D. and Hurihurun. P.V. (1911%Int. J, Ra diut. WI. 48.691-700. [48) Ladon. S,A, (1990) Hculth Phyr. 59, 15-22. (491 Sxittroit3ki. T.P. ;lad Nathan. CF. (1991) Cancer Ra. 51. 791798. (50) US Dcparmcn~ nf Hcal~b Education und Wclfarc. Smokiny und Hculth, A Report of the Surgeon Gcncr;tl (IW) RHEW Publication, No, (PHS) 79.SOW, p. 3. 1
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WI P1dwww ‘I’., Kwke, M.. Ksdumu. M. und Nugu~u.C. (lN5) NwJrc 3 14, 462a4. I521 Prsar. W-A. (19871 Ur. f. Cunccr 55 (arppl. VIII), 1%.23. IS31 Lcrukkrsw. POnndTugwon.C, (1992)Chctwl3iol. 11%81. l97.208. 1541Cifonc. M.A. und Fidlcr. I.J. (1961) Proe. Nutl. Awd. Sci. USA 78.6949-4952. IS51 Mcyidish, T. und Mazurck. N.A. (1989) Netwe 342, 807-till.
