[CANCER RESEARCH 51. 5837-5842, November 1. 1991]

Nickel(H)- and Cobalt(II)-dependent Damage by Hydrogen Peroxide to the DNABases in Isolated Human Chromatin1

Zeena Nackerdien, Kazimierz S. Kasprzak, Govind Rao, Barry Halliwell,2 and Mirai Dizdaroglu3

Chemical Science and Technolog)' Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 [Z. N., M. D.¡;Departmentof Radiotherapy, University of Stellenbosch, Tygerberg, South Africa [Z. N.]; Chemical and Biochemical Engineering, University of Maryland Baltimore Countyand Medical Biotechnology Center, Maryland Biotechnology Institute, Baltimore, Maryland 21228 [G. R.J; Laboratory of Comparative Carcinogenesis, NationalCancer Institute, FCRDC, Frederick, Maryland 21702 ¡K.S. K.J; and Pulmonary Medicine, University of California Davis Medical Center, Sacramento,California 95817 [B. H.J

ABSTRACT

Nickel compounds are known to be carcinogenic to humans andanimals. Cobalt compounds produce tumors in animals and are probablycarcinogenic to humans. The mechanisms of the carcinogenicity of thesemetal compounds, however, have remained elusive. In the present work,we have investigated the ability of Ni(II) and <'<i(11) ions in the presence

of 11>()jto cause chemical changes in DNA bases in chromatin extractedfrom cultured cells of human origin. Eleven modified DNA bases inchromatin were identified and quantitated by the use of gas chromatog-raphy-mass spectrometry. 2-Hydroxyadenine (isoguanine), which has notpreviously been shown to occur in DNA or chromatin, was also identified.Products identified were typical hydroxyl radical-induced products ofDNA bases, suggesting that the hydroxyl radical was involved in theirformation. This idea was supported by partial inhibition of productformation by typical scavengers of hydroxyl radical. Partial inhibition ofproduct formation indicated a possible "site-specific" formation of hydroxyl radical by unchelated Ni(II) and ('o(II) ions bound to chromatin.

Although treatment of chromatin for l h with Co(II)/H2O2 caused formation of significant amounts of products, treatment with Ni(ll)/lI..O.required incubation times of more than 5 h and an increase in NilII)concentration before increases in product amounts above backgroundlevels became detectable. In both cases, ascorbic acid did not increaseproduct yields. Glutathione at a physiologically relevant concentrationhad little overall effect on DNA base modification. Superoxide dismutaseincreased the yields of most products. Chelation of Ni(II) and ( o(II ) ionswith EDTA almost completely inhibited product formation. Nil III in thepresence of 11;<>2produced greater base damage to the DNA in chromatinthan to isolated DNA, unlike other metal ions tested. DNA damage inchromatin caused by Ni(II) and Co(II) ions in the presence of H2O2 maycontribute to the established genotoxicity and carcinogenicity of thesemetal ions.

INTRODUCTION

Oxygen-derived species such as the Superoxide radical(O2^),4 H2O2, and the hydroxyl radical (OH) have been impli

cated in the etiology of many human diseases including cancer(reviewed in Ref. l). Thus, an increased production of oxygen-derived species within cells frequently leads to DNA damage

Received 5/23/91; accepted 8/15/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by the Office of Health and Environmental

Research, Office of Energy Research, US Department of Energy, Washington,DC. Z. N. received support from the South African Medical Research Council.G. R. was supported by the National Science Foundation (EET-8808775). B. H.is the recipient of research support from the Medical Research Council and theArthritis and Rheumatism Council. United Kingdom.

1 Permanent address: Biochemistry Department, University of London King's

College, Strand Campus, London WC2R 2LS, United Kingdom.3To whom requests for reprints should be addressed.'The abbreviations used are: O¡",Superoxide radical; OH, hydroxyl radical;

GC/MS-SIM, gas chromatography-mass spectrometry with selected-ion monitoring; 5-OH-5-Me-Hyd, 5-hydroxy-5-methylhydantoin; 5-OH-Hyd, 5-hydroxy-hydantoin; 5-OHMe-Ura, 5-(hydroxymethyl)uracil; 5,6-diOH-Cyt, 5,6-dihydrox-ycytosine; FapyAde, 4,6-diamino-5-formamidopyrimidine; 8-OH-Ade, 8-hydrox-yadenine; 2-OH-Ade, 2-hydroxyadenine (isoguanine); FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8-OH-Gua, 8-hydroxyguanine; DMSO, dimethyl sulfoxide; SOD, Superoxide dismutase.

by a variety of mechanisms (reviewed in Ref. 2), and suchspecies can probably both initiate and promote cancer (2, 3).However, neither O2" nor H2O2 reacts chemically with DNA

unless metal ions are present in the system (2,4, 5). By contrast,highly reactive OH attacks all constituents of DNA producinga multiplicity of chemical changes in the deoxyribose, pyrimi-dines, and purines (reviewed in Ref. 6). DNA-protein crosslinks also result from OH attack upon nucleoprotein (reviewedin Ref. 7). Indeed, the pattern of chemical changes produced inpyrimidine and purine bases when DNA is exposed to OH isso characteristic that it can be held to be diagnostic for attackby OH, since no other species so far examined has producedsuch a range of chemical modifications of DNA bases (reviewedin Refs. 2 and 8). For example, examination of chemicalchanges in the DNA bases has been used to show that damageto DNA by a Cu(II)-o-phenanthroline complex in the presenceof a reducing agent probably involves OH (9), whereas OH doesnot contribute significantly to DNA base damage by the bleo-mycin/Fe(III)/ascorbic acid system (10).

A number of transition metal ions can catalyze OH formationfrom O2" and H2O2 and, thus, can induce DNA damage in the

presence of these oxygen-derived species. They include ironions and copper ions (4, 11-13). Nickel in many physicochem-ical forms is well established to be carcinogenic to humans andanimals (14-17). However, the mechanisms involved in theprocess of tumor production remain elusive. In mammaliancells, nickel affects the genetic material, producing sister-chro-matid exchanges and chromosomal aberrations (18, 19). Thereis evidence for binding of Ni(II) to cell nuclei (20, 21) and forinduction by Ni(II) of DNA strand breaks and DNA-proteincross-links (20, 22-25). However, Ni(II) alone causes no damage to isolated DNA (26), and the relatively weak interactionsbetween Ni(II) and DNA are unlikely to be responsible forgenotoxic effects in cells exposed to Ni(II) (25). Thus, it hasbeen proposed that Ni(II) reacts with endogenous H2O2 in cellsto form OH, which causes DNA damage (26, 27). Recent invitro studies have indicated the formation of OH in reactionsof Ni(II) and Ni(II)-peptide complexes with H2O2 (26-29). Onthe other hand, studies of the effects of OH scavengers gaveequivocal results (26). When OH is generated by reaction ofH2O2 with transition metal ions bound to the DNA, it is oftendifficult to completely protect the DNA from OH attack byadding OH scavengers because of the possible "site-specific"generation of OH (11-13, 30, 31).

Cobalt is also thought to be a carcinogenic metal (reviewedin Ref. 32). The genotoxic effects of cobalt salts in human andanimal cell cultures and their carcinogenicity in humans andanimals have recently been reviewed in detail (33). The production of a number of other biological dysfunctions by cobaltcompounds in vitro and in vivo has also been reported (23, 34-38). On the basis of the evidence available, it has been suggestedthat cobalt compounds be classified as probable chemical carcinogens for humans (33). The mechanisms underlying cobalt

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Ni(Il)- AND Co(II)-DEPENDENT H¡O¡DAMAGE TO HUMAN CHROMATIN DNA

toxicity have not been established, but oxygen-derived speciesmay be involved (39). It has been proposed that oxidation ofsome organic compounds by cobalt complexes in the presenceof alkyl hydroperoxides, oxygen, or hydrogen peroxide involvesoxygen-centered free radicals and proceeds via a Haber-Weissmechanism (40-42). It has been suggested that Co(II) reactswith H2O2 to form OH (38, 41, 43), but this view has beenquestioned on the basis of electron spin resonance studies (44).

In the present work, we have investigated the ability ofmixtures of HjO^ with Ni(II) or Co(II) to cause chemicalchanges in the DNA bases in isolated human chromatin. Thetechnique of GC/MS-SIM was used to measure these base

modifications.

MATERIALS AND METHODS

Materials.9 Histones HI, H2A, H2B, H3, and H4 were purchased

from Boehringer Mannheim. Chelex 100 resin (200 to 400 mesh) andreagents for electrophoresis were purchased from Bio-Rad. Acetonitrileand bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlo-rosilane were obtained from Pierce Chemical Company. Dialysis membranes with a molecular weight cut-off of 3500 were purchased fromFisher Scientific Company. Formic acid (88%) was obtained fromMallinckrodt. Fetal bovine serum, RPMI Medium R5507, L-glutamine,EDTA, DMSO, mannitol, ascorbic acid, 5-OHMe-Ura, isobarbituricacid (5-hydroxyuracil), FapyAde, 6-azathymine, 8-azaadenine, isogua-nine (2-OH-Ade), glutathione, catatase (type C-40, thymol free), copper,zinc-superoxide dismutase, and calf thymus DNA were purchased fromSigma Chemical Co. Penicillin and streptomycin were from Gibco. 2-Amino-6,8-dihydroxypurine (8-OH-Gua) was from Chemical Dynamics Corporation. 5,6-Dihydroxyuracil (dialuric acid) was obtained fromAmerican Tokyo Kasei, Inc. 5-Hydroxy-5-methylhydantoin was a giftfrom Dr. W. F. Blakely, Armed Forces Radiobiology Research Institute,Bethesda, MD. The synthesis of thymine glycol, 8-OH-Ade, andFapyGua was described elsewhere (45, 46). There was no referencecompound available for 5-OH-Hyd. The gas Chromatographie retentiontime and the mass spectrum of this compound were obtained using asample of cytosine, which was -y-irradiated in N2O/O2-saturated

aqueous solution.Cell Culture and Isolation of Chromatin. The cells used for chromatin

isolation were K562 human cells (courtesy of Dr. S. A. Akman, City ofHope National Medical Center, Duarte, CA). Suspension cultures ofthis cell line were incubated at 37°Cunder an atmosphere of 3% COi

mixed with room air. The growth medium consisted of RPMI mediumsupplemented with 10% fetal bovine serum, L-glutamine (4 mM), penicillin (50 units/ml), and streptomycin (50 Mg/ml). Isolation of chromatin was performed as described previously (47). Chromatin wasobtained in 1 mM Tris-buffer (pH 7.4) and then dialyzed extensivelyagainst 1 mM phosphate buffer (pH 7.4) containing 0.2 mM EDTA,followed by extensive dialysis against 1 mM phosphate buffer (pH 7.4)treated with Chelex resin. Solutions of calf thymus DNA were treatedwith EDTA and Chelex resin in the same manner. Dialyses were carriedout at 4°C.After dialysis, chromatin was homogenized briefly with a

few strokes in a glass homogenizer. Chromatin was characterized asdescribed previously (47). The ratio of the amount of protein to that ofDNA was 1.8. The RNA content of chromatin was £5%of the amountof DNA. The chromatin exhibited the following spectral characteristics:A258/A28o= 1-62; A258/A23o=1.12; A258/A32o= 11.4; and A(maximum)/A(minimum) = 1.46. The protein components of chromatin wereanalyzed by gel electrophoresis as described previously (47).

Treatment of Chromatin. Reaction mixtures contained the followingcompounds, where appropriate, in a final volume of 1.2 ml of 1 mMphosphate buffer (pH 7.4): chromatin (0.12 mg of DNA/ml); NiCl2 (25

*Certain commercial equipment or materials are identified in this paper inorder to specify adequately the experimental procedure. Such identification doesnot imply recommendation or endorsement by the National Institute of Standardsand Technology, nor does it imply that the materials or equipment identified arenecessarily the best available for the purpose.

MMor 100 MM);CoSO4 (25 MMor 100 MM);EDTA (120 MM);H2O2 (2.8mM or 10 mM); ascorbic acid (100 MM);glutathione (1 mM); mannitol(50 mM); DMSO (50 HIM);catalase (1100 units/ml); and SOD (200units/ml). One unit of catalase decomposes 1 MHIO!of H2O2/min at pH7.0 at 25°C,under the conditions given in the Sigma catalog. Units of

SOD were as defined by the cytochrome c assay (48). In experimentswith EDTA, NiCl2 and CoSO4 were mixed with EDTA (120 MM)priorto addition to reaction mixtures. Chelex-treated phosphate buffer (1mM, pH 7.4) was used for all dilutions. Mixtures were incubated at37°Cfor 1 to 24 h. After incubation, 0.5 nmol of 6-azathymine and 2

nmol of 8-azaadenine were added to aliquots of chromatin samplescontaining 0.12 mg of DNA. The samples were immediately frozen inliquid nitrogen and then lyophilized.

Hydrolysis with formic acid of lyophilized samples of chromatin,trimethylsilylation of hydrolysates, and analysis of trimethylsilylatedhydrolysates by GC/MS-SIM were performed as described previously(47).

RESULTS

The protein components of the isolated chromatin wereexamined by gel electrophoresis using commercially availablehistones as reference compounds. Fig. 1 illustrates typical gelelectrophoretic patterns of the proteins of the isolated chromatin and commercial histones. The patterns of histones inFig. 1 are similar to those of histones published previously (47,49). Fig. 1 shows that histones HI, H2A, H2B, H3, and H4were present in isolated chromatin.

Trimethylsilylated hydrolysates of chromatin were analyzedby GC/MS-SIM. The use of this technique for measurement offree radical-induced base products in DNA and in chromatinhas been described previously (13, 46, 47, 50, 51). Fig. 2illustrates a representative chromatogram with selected-ion current profiles, which were obtained during the GC/MS-SIManalysis of the trimethylsilylated hydrolysate of a Ni(II)/H2O2-treated chromatin sample. Peak identification is given in the

HI

H3H2BH2A

H4

Fig. I. Gel electrophoretic patterns of isolated human chromatin (Lane I) andcommercial histones HI, H3, H2B, H2A, and H4 (Lane 2).

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N¡(II)-AND Codn-DEPENDENT H¡OjDAMAGE TO HUMAN CHROMATIN DNA

figure legend. In addition to the base products found in chro-matin in our previous publications (13, 47), 2-hydroxyadenine(isoguanine) was also observed in the present work.

Effect of Co(II) Ions. The yields of DNA base products formedin isolated chromatin by treatment with the Co(II)/H2O2 systemare given in Table 1. DNA in chromatin already containedmeasurable amounts of various modified bases (Table 1), asdescribed in our earlier papers (13,47). Treatment of chromatinwith Co(II) alone or H2O2 alone did not cause any significantincreases in the background amounts of the products. Treatment of chromatin with Co(II)/H2O2 for l h caused significantincreases in the amounts of all base products (Table 1). Cytosineglycol, formamidopyrimidines, and 8-hydroxypurines were themajor products. Ratios of yields to background levels variedbetween «2(5-OH-Hyd) and ~18 (thymine glycol). Addition

of ascorbic acid (100 MM)had no effect upon product yields.The OH scavengers mannitol and DMSO at high concentrations (50 mM) provided significant inhibition of product for

mation (Table 1). However, yields of products were still higherthan background levels (Table 1). Glutathione at a physiologically relevant concentration (1 HIM) caused only a moderatedecrease in the yields of some products (Table 1). On the otherhand, the yields of cytosine glycol and FapyGua were doubledby glutathione (Table 1). Addition of SOD to Co(II)/H2O2 didnot reduce product yields (Table 1). On the contrary, an «2-fold increase in product yields was observed. Chelation of Co(II)(25 ßM)with EDTA (120 /¿M)substantially inhibited productformation. For example, the yields of 8-OH-Gua, 8-OH-Ade,and FapyAde given in Table 1 were decreased to 0.59 ±0.06,0.24 ±0.036, and 0.17 ±0.005 nmol/mg of DNA, respectively(also compare these values with those of column 1 in Table 1).

Effect of Ni(II) Ions. Unlike the case of Co(II), treatment ofchromatin with Ni(II) (25 //M)/H2O2 (2.8 mM and up to 10HIM) produced no increase in the amounts of modified bases.Therefore, the amount of Ni(II) in the reaction mixtures wasincreased to 100 UM, and a time course study was undertaken.

70000-60000-50000-5

40000-c',-a

',§

30000-è20000-10000-0.41IJ*3ÄA,

J65"A-3451215107ÃŒJ

9K11sJ-^

—¿�131"~>^JlLA.JU6

7 8 9 10 1112T1me (min. )

Fig. 2. Selected-ion current profiles obtained during the GC/MS-SIM analysis of a trimethylsilylated hydrolysate of chromatin treated with Ni(II) (100 /iM)/H2O2(2.8 mM) for 24 h. For experimental details, see "Materials and Methods." The temperature of the oven of the gas Chromatograph was programmed from 150-260*Cat a rate of 8'C/min after 2 min at 150'C. The amount of an aliquot of DNA in chromatin injected onto the column was approximately 0.4 /jg. Peaks (ion): I, 6-azathymine (m/z 256) (internal standard); 2, 5-hydroxy-5-methylhydantoin (m/z 331); 3, 5-hydroxyhydantoin (m/z 317): 4, 5-hydroxyuracil (m/z 329); 5, 5-(hydroxymethyl)uracil (m/z 358): 6, 5-hydroxycytosine (m/z 343): 7, c/'j-thymine glycol (m/z 259); 8, 5,6-dihydroxyuracil (m/z 417); 9, frans-thymine glycol (m/z

259); 10, 8-azaadenine (m/z 265) (internal standard); //, 4,6-diamino-5-formamidopyrimidine(m/z 354); 12, 8-hydroxyadenine (m/z 352); 13, 2-hydroxyadenine (mlz 352); 14, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (m/z 442); IS, 8-hydroxyguanine (m/z 441). The m/z 441 ion is an isotope-ion of m/z 440, the abundanceof which was approximately 2.5 times that of m/z 441. The m/z 440 ion |(M - 15)+ ion] was used for quantitation of 8-hydroxyguanine. It should be noted that 5-hydroxyuracil (Peak 4) and 5-hydroxycytosine (Peak 6) arise by acid-induced modification of cytosine glycol. Similarly. 5,6-dihydroxyuracil (Peak 8) is produced byacid-induced deamination of 5,6-dihydroxycytosine. 5-Hydroxy-5-methylhydantoin (Peak 2) and 5-hydroxyhydantoin (Peak 3 ) are believed to result from acid-inducedmodification of 5-methyl-5-hydroxybarbituric acid and 5,6-dihydroxycytosine, respectively.

Table 1 Yields (nmol/mg of DNA) of DNA base products formed in chromatin by treatment with the Co(lI)/H2O2 system (25 tíM/208mM, respectively, for I h)Treatment"

Product5-OH-5-Me-Hyd5-OH-Hyd5-OHMe-UraCytosine

glycolThymineglycol5,6-diOH-CytFapyAde8-OH-Ade2-OH-AdeFapyGua8-OH-Gua10.074

±0.043*0.250

±0.0250.024±0.0030.082±0.0130.01

7±0.0020.030±0.0030.1

15±0.0280.113±0.0350.078

±0.0160.471±0.0730.526

±0.16520.505

±0.081C0.548±0.062C0.227±0.041C1.87±0.45C0.289

±0.002'0.344±0.044'0.895±0.255e1.

02 ±0.29e0.177 ±0.056e1.86

±0.53'5.71±1.99r30.390

±0.035'0.652

±0.0690.080±0.010''0.484

±0.204''0.238

±0.1200.077±0.038''0.380

±0.113''0.290±0.042''0.1

89±0.0301.73

+ 0.59''40.218

±0.011''0.380±0.092''0.081

±0.007''0.478±0.145''0.088

±0.039''0.095±0.014''0.318

±0.055''0.293±0.050*0.100+0.009''1.09+0.232.40±0.57*50.2780.3190.1703.750.2250.3020.7350.5130.1083.760.046''0.061''0.044''0.24*0.0010.0230.1080.031*0.0170.374''4.78±0.14561.38

±0.31''1.03+0.06''0.485+ 0.134*3.01

±0.48*0.409

±0.1560.636+0.076*1.47+0.044*2.20+0.066*0.297±0.067*2.99+0.31*12.20+2.62*

" 1, chromatin; 2, chromatin/Co(II)/H2O2; 3, chromatin/Co(II)/H2O2/mannitol (50 mM); 4, chromatin/Co(II)/H2O2/DMSO (50 mM); 5, chromatin/Co(II)/H2O2/

glutathione (1 mM); 6, chromatin/Co(II)/H2O2/SOD (200 units/ml).* Mean ±SD (n = 3).' Values significantly different from those of Column 1 (P < 0.05).* Values significantly different from those of Column 2 (P < 0.05).

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Ni(II)- AND Co(II)-DEPENDENT H2O2 DAMAGE TO HUMAN CHROMATIN DNA

Again, the treatment of chromatin with Ni(II) (100 ßM)andH2O2 (2.8 ITIM)for l h produced no significant rises in theamounts of the modified bases over background levels. However, treatment for 5 to 24 h increased the amounts of allproducts. As representative examples, Fig. 3 illustrates thedependence of the yields of two modified bases on the treatmenttime. DNA base damage caused by Ni(II)/H2O2 increased steadily up to 24 h of treatment. For the rest of the experiments withNi(II), a treatment time of 24 h was used. Results obtained withNi(II) (100 fiM) are shown in Table 2. Ni(II) alone causedsignificant increases in the amounts of some, but not all, products (Table 2). Addition of catalase (1100 units/ml) with theNi(II) caused no change in the product yields shown. Treatmentwith H2O2 alone for 24 h also raised the background amountsof several products (Table 2). Since H2O2 does not react withDNA (4, 5), this may indicate the presence of some remainingmetal ions which were not completely removed by the experimental procedures used. Ni(II)/H2O2 markedly increased product yields over the levels observed with Ni(II) alone or H2O2alone. The highest proportional increase (=6-fold) was in theyields of 8-hydroxypurines (Table 2). Cytosine glycol, forma-midopyrimidines, and 8-hydroxypurines were the major products, as in the case with Co(II)/H2O2. As for Co(II), the additionof ascorbic acid (100 ^M) to Ni(II)/H2O2 had no significanteffect on product yields. DMSO partially decreased the yieldsof some products up to 60% (Table 2). Nevertheless, productyields were still much higher than background levels. Mannitolhad a similar effect (data not shown). Glutathione at 1 ITIMhadan inhibitory effect on most of the products, although the yieldof FapyGua was doubled (Table 2), in analogy to the resultsobtained with Co(II). The presence of SOD in the reaction

mixture did not inhibit product formation. On the contrary, anœ2-foldincrease in the yields of 5-OHMe-Ura, cytosine glycol,FapyAde, 8-OH-Ade, and 2-OH-Ade was observed (Table 2).Chelation of Ni(II) (100 ¿ZM)with EDTA (120 MM)caused amarked decrease in the yields of all products. For example, theyields of 8-OH-Gua, 8-OH-Ade, and thymine glycol given in

Table 2 were decreased to 0.80 ±0.08, 0.53 ±0.18, and 0.31±0.09, respectively (also compare these values with thoseelsewhere in Table 2).

Effect on Calf Thymus DNA. Table 3 illustrates the resultsobtained with calf thymus DNA under the same conditions andusing the same amount of DNA as in chromatin. Treatment ofcalf thymus DNA with Co(II) (25 MM)/H2O2for l h producedsignificant increases in the amounts of modified bases (Table3). Except for the yields of formamidopyrimidines, the productyields in calf thymus DNA were higher than those in chromatin(compare Table 1 with Table 3). Treatment of calf thymus DNAwith Ni(II) (100 ¿iM)/H2O2for 24 h significantly increased theamounts of products (Table 3). The product yields obtained incalf thymus DNA with Ni(H)/H2O2 treatment were lower (except for the yield of thymine glycol) than those in chromatin(compare Table 2 with Table 3).

DISCUSSION

The DNA base products identified in isolated human chromatin in the present work are typical products arising fromreactions of OH with the DNA bases (reviewed in Refs. 6, 8,and 52). This pattern of products suggests that reaction ofCo(II) or Ni(II) with H2O2 produces OH, which attacks theDNA bases. Partial inhibition of base modification by typical

Fig. 3. Dependence of the yields of 8-OH-Ade and 8-OH-Gua on the treatment time. A,chromatin; •¿�,chromatin/H2O2; D, chromatin/Ni(II)/H2O2. Points, mean; bars, SD.

8-OH-Ade

-~ 304Za•¿�5

8-OH-Gua

time (h) tima (h)

Table 2 Yields (nmol/mg of DNA) of DNA base products formed in chromatin by treatment with the Ni(II)/H2O2 system (¡00nM/2.8 mM, respectively, for 24 h)Treatment"

Product5-OH-5-Me-Hyd5-OH-Hyd5-OHMe-UraCytosine

glycolThymineglycol5,6-diOH-CytFapyAde8-OH-Ade2-OH-AdeFapyGua8-OH-Gua10.1

76±0.052*1.05

±0.0420.097±0.0220.1

53±0.0060.031±0.0040.079±0.0160.340±0.0490.277±0.0370.140+0.0051.88

±0.0741.14±0.10820.407

±0.062f1.33

±0.1230.144±0.015C0.1

46±0.0420.084±0.0490.185±0.028C0.308

±0.0560.591±0.152'0.419

±0.047'2.11

±0.0302.73±0.406f30.538

±0.074'1.22

±0.0640.204±0.097C1.04±0.133C0.578

±0.095C0.151

±0.0530.728±0.009'0.646±0.024C0.374±0.009C2.55+0.3154.62±0.131'41.97±

1.09¿1.69

±0.820.632±0.127¿3.37

+ 0.716J1.02±0.178''0.597±O^SO*2.33

±0.169''4.02±0.649''0.567

±0.0576.39±0.789''30.2±1.77'50.796

±0.153°1.33+0.2040.631

±0.2112.99+0.3960.603±0.2090.1

78±0.074'1.45

±0.4323.29+0.100.653+0.1203.

14±0.658'13.8±1.34'61.14

±0.310'0.829+0.270'0.378±0.049'2.00

±0.6290.544±0.152'0.668+0.0541.44±0.217'0.508±0.052'0.193+0.029'14.7±3.89'18.9±2.67'71.69

±0.1771.74±0.1261.53

+0.212'5.70±0.637'1.14+0.0360.550+0.0073.41

±0.403'9.28±1.06'0.971

±0.132'5.05

±0.62830.3+ 3.10

•¿�I, chromatin; 2, chromatin/Ni(II); 3, chromatin/H2O2; 4, chromatin/Ni(II)/H2O2; 5, chromatin/Ni(II)/H2O2/DMSO (50 «M);6, chromatin/Ni(II)/H2O2/

glutathione (1 ITIM);7, chromatin/Ni(H)/H2O2/SOD (200 units/ml).* Mean ±SD (n = 3).c Values significantly different from those of Column 1 (P< 0.05).d Values significantly different from those of Columns 2 and 3 (P < 0.05).' Values significantly different from those of Column 4 (P< 0.05).

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Ni(II)- AND Co(II)-DEPENDENT H¡O:DAMAGE TO HUMAN CHROMATIN DNA

Table 3 Yields (nmol/mg of DNA) of base products formed in calf thymus DNA by treatment with Co(Il)/H2O2 and Ni(II)/H2O, systemsTreatment"

Product5-OH-5-Me-Hyd5-OH-Hyd5-OHMe-UraCytosine

glycolThymineglycol5.6-diOH-CytFapyAde8-OH-Ade2-OH-AdeFapyGua8-OH-Gua10.129

±0.017*0.079

±0.0120.032±0.0060.209±0.0300.556±0.1790.006±0.0020.164

±0.0250.088±0.0100.039±0.0070.242±0.0530.389±0.04720.217

±0.0190.104±0.0210.061

±0.0080.898±0.0590.932±0.0990.012

±0.0020.235±0.0390.166

±0.0130.039±0.0070.352±0.0740.996±0.18331.66±0.12r0.702

±0.076'0.568±0.062'3.69±0.25'3.34±0.03'0.580±0.09\'0.667±0.042'2.15

±0.286'0.162±0.01'0.789

±0.138'7.37±0.87'41.42±0.30r1.24

±0.47'0.296±0.015'1.29

±0.0975.01±1.09'0.211

±0.033'0.613±0.059'3.08

±0.55'0.148±0.021'0.717

±0.080'7.23±0.827'

' 1, DNA (24 h); 2, DNA/H2O2 (24 h); 3, DNA/Co(II) (25 »M)/H2O,(I h); 4, DNA/Ni(II) (100 MM)/H2Oj (24 h).' Mean ±SD (n = 3).' Values significantly difTerent from those of Column 2 (P < 0.05).

OH scavengers is consistent with this view. The failure of chromatin by the presence of SOD in mixtures containingscavengers to prevent DNA damage in chromatin completelymight be due to "site-specific" formation of OH (11, 30, 31).

Unchelated metal ions might bind to DNA and/or to othercomponents of chromatin and cause formation of OH in closeproximity to DNA. Thus, OH produced this way may reactwith DNA to a greater extent than with scavengers or berelatively inaccessible to scavengers.

In contrast to Co(II) alone, Ni(II) alone caused significantrises in the background amounts of modified DNA bases inchromatin. This may be due to the ability of complexes of Ni(II)with certain peptide sequences in chromatin to generate freeradicals in the presence of oxygen, as described previously (25,S3,54). Failure of catatase to inhibit the damage by Ni(II) aloneindicates that H2O2 may not be required for such reactions. Thefact that the yields of products generated by Ni(II)/H2O2 werehigher in chromatin than in calf thymus DNA may indicate theenhancement of free radical production by Ni(II)-peptide (orprotein) complexes and is of special interest in relation to theestablished carcinogenicity of this metal. On the other hand,Co(II)/H2O2 caused more damage in both chromatin and calfthymus DNA than did Ni(II)/H2O2. In the latter case, a substantial increase in Ni(II) concentration and in treatment timewas necessary to produce significantly higher amounts than thebackground amounts of base products in chromatin.

Addition of ascorbic acid had little effect on product yields.By contrast, ascorbic acid greatly stimulates DNA base modification produced by Cu(II)/H2O2 or Fe(III)/H2O2 (13). Thus,contamination of the chromatin or of the added Co(II) or Ni(II)with Cu(II) or Fe(III) is ruled out as explanation of the resultsobtained in the present work. The presence of glutathione inreaction mixtures at a physiologically relevant concentrationcaused no marked inhibition of DNA base damage. The observed increase in the yield of FapyGua in the case of bothCo(II) and Ni(II) could result from increased reduction byglutathione of the C-8 OH adduct radical that is formed fromaddition of OH to the C-8 position of guanine. The site specificity of OH~ generation may account for the inability of

glutathione to markedly inhibit the DNA damage. These resultssuggest that glutathione may not be able to prevent the DNAdamage mediated by bound-metal ions in vivo and are consistentwith previous findings (55, 56).

The inability of SOD to inhibit the product formation suggests that O2" was not required in generation of OH by Co(II)/

H2O2 or Ni(II)/H2O2. The observed increase in product yieldsby addition of SOD to Co(II)/H2O2 or to Ni(II)/H2O2 mayindicate additional generation of OH in these systems by anunknown mechanism. Stimulation of DNA damage in isolated

Cu(II)/H2O2 or Fe(III)/H2O2 has also been observed previously(13,57).

Inhibition of product formation by chelation of Co(II) andNi(II) with EDTA is analogous to the results obtained withCu(II), but in contrast to those obtained with Fe(III) undersimilar reaction conditions (13, 57). The inability of Co(II)/EDTA to produce DNA base modification is in agreement withthe results reported by Kadiiska et al. (44).

In conclusion, Co(II) and Ni(II) ions in the presence of H2O2cause formation of typical OH-induced products of DNA basesin isolated human chromatin. Partial product inhibition bytypical scavengers of OH supports the idea of the involvementof OH in product formation. Substantial quantitative differences exist between the effects of Co(II) and Ni(II). DNA basedamage in chromatin mediated by Co(II) and Ni(II) may contribute to the established genotoxicity and carcinogenicity ofthese metal ions.

ACKNOWLEDGMENTS

We are grateful to Dr. S. A. Akman of the City of Hope NationalMedical Center, Duarte, CA, for a sample of the K562 cell line.

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1991;51:5837-5842. Cancer Res   Zeena Nackerdien, Kazimierz S. Kasprzak, Govind Rao, et al.   Peroxide to the DNA Bases in Isolated Human ChromatinNickel(II)- and Cobalt(II)-dependent Damage by Hydrogen

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