[CANCER RESEARCH 51. 5837-5842, November 1. 1991] Nickel(H)- and Cobalt(II)-dependent Damage by Hydrogen Peroxide to the DNA Bases 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.¡;Department of Radiotherapy, University of Stellenbosch, Tygerberg, South Africa [Z. N.]; Chemical and Biochemical Engineering, University of Maryland Baltimore County and Medical Biotechnology Center, Maryland Biotechnology Institute, Baltimore, Maryland 21228 [G. R.J; Laboratory of Comparative Carcinogenesis, National Cancer 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 and animals. Cobalt compounds produce tumors in animals and are probably carcinogenic to humans. The mechanisms of the carcinogenicity of these metal compounds, however, have remained elusive. In the present work, wehave investigated the ability of Ni(II) and <'<i(11)ions in the presence of 11>()jto cause chemical changes in DNA bases in chromatin extracted from cultured cells of human origin. Eleven modified DNA bases in chromatin were identified and quantitated by the use of gas chromatog- raphy-mass spectrometry. 2-Hydroxyadenine (isoguanine), which has not previously been shown to occur in DNA or chromatin, was also identified. Products identified were typical hydroxyl radical-induced products of DNA bases, suggesting that the hydroxyl radical was involved in their formation. This idea was supported by partial inhibition of product formation by typical scavengers of hydroxyl radical. Partial inhibition of product formation indicated a possible "site-specific" formation of hy droxyl radical by unchelated Ni(II) and ('o(II) ions bound to chromatin. Although treatment of chromatin for l h with Co(II)/H2O2 caused for mation 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 background levels became detectable. In both cases, ascorbic acid did not increase product yields. Glutathione at a physiologically relevant concentration had little overall effect on DNA base modification. Superoxide dismutase increased the yields of most products. Chelation of Ni(II) and ( o(II ) ions with EDTA almost completely inhibited product formation. Nil III in the presence of 11;<>2produced greater base damage to the DNA inchromatin than to isolated DNA, unlike other metal ions tested. DNA damage in chromatin caused by Ni(II) and Co(II) ions in the presence of H2O2 may contribute to the established genotoxicity and carcinogenicity of these metal 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 in accordance 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 the Arthritis and Rheumatism Council. United Kingdom. 1 Permanent address: Biochemistry Department, University of London King's College, Strand Campus, London WC2R 2LS, United Kingdom. 3 To 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 moni toring; 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, di methyl sulfoxide; SOD, Superoxide dismutase. by a variety of mechanisms (reviewed in Ref. 2), and such species 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 producing a multiplicity of chemical changes in the deoxyribose, pyrimi- dines, and purines (reviewed in Ref. 6). DNA-protein cross links also result from OH attack upon nucleoprotein (reviewed in Ref. 7). Indeed, the pattern of chemical changes produced in pyrimidine and purine bases when DNA is exposed to OH is so characteristic that it can be held to be diagnostic for attack by OH, since no other species so far examined has produced such a range of chemical modifications of DNA bases (reviewed in Refs. 2 and 8). For example, examination of chemical changes in the DNA bases has been used to show that damage to DNA by a Cu(II)-o-phenanthroline complex in the presence of a reducing agent probably involves OH (9), whereas OH does not 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 formation from O2" and H2O2 and, thus, can induce DNA damage in the presence of these oxygen-derived species. They include iron ions and copper ions (4, 11-13). Nickel in many physicochem- ical forms is well established to be carcinogenic to humans and animals (14-17). However, the mechanisms involved in the process of tumor production remain elusive. In mammalian cells, nickel affects the genetic material, producing sister-chro- matid exchanges and chromosomal aberrations (18, 19). There is evidence for binding of Ni(II) to cell nuclei (20, 21) and for induction by Ni(II) of DNA strand breaks and DNA-protein cross-links (20, 22-25). However, Ni(II) alone causes no dam age to isolated DNA (26), and the relatively weak interactions between Ni(II) and DNA are unlikely to be responsible for genotoxic effects in cells exposed to Ni(II) (25). Thus, it has been proposed that Ni(II) reacts with endogenous H2O2 in cells to form OH, which causes DNA damage (26, 27). Recent in vitro studies have indicated the formation of OH in reactions of Ni(II) and Ni(II)-peptide complexes with H2O2 (26-29). On the other hand, studies of the effects of OH scavengers gave equivocal results (26). When OH is generated by reaction of H2O2 with transition metal ions bound to the DNA, it is often difficult to completely protect the DNA from OH attack by adding OH scavengers because of the possible "site-specific" generation of OH (11-13, 30, 31). Cobalt is also thought to be a carcinogenic metal (reviewed in Ref. 32). The genotoxic effects of cobalt salts in human and animal cell cultures and their carcinogenicity in humans and animals have recently been reviewed in detail (33). The produc tion of a number of other biological dysfunctions by cobalt compounds in vitro and in vivo has also been reported (23, 34- 38). On the basis of the evidence available, it has been suggested that cobalt compounds be classified as probable chemical car cinogens for humans (33). 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