Interactions of Nickel (II) with Histones: Enhancement of 2

Marios Mylonas, Gerasimos Malandrinos, John Plakatouras, Nick Hadjiliadis, Kazimierz S. Kasprzak, Artur Krȩżel, and Wojciech Bal. Chemical Research ...
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Chem. Res. Toxicol. 1996, 9, 535-540

535

Interactions of Nickel(II) with Histones: Enhancement of 2′-Deoxyguanosine Oxidation by Ni(II) Complexes with CH3CO-Cys-Ala-Ile-His-NH2, a Putative Metal Binding Sequence of Histone H3 Wojciech Bal,*,†,‡ Jan Lukszo,§ and Kazimierz S. Kasprzak† Laboratory of Comparative Carcinogenesis, National Cancer Institute, FCRDC, Frederick, Maryland 21702, and Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, Rockville, Maryland 20852 Received September 12, 1995X

Studies of 2′-deoxyguanosine oxidation by hydrogen peroxide in the presence of CH3COCys-Ala-Ile-His-NH2 (CAIH) and/or NiCl2 have been carried out in 100 mM phosphate buffer (pH 7.4) at 37 °C. The dimeric CAIH oxidation product, CAIH disulfide, and its weak, octahedral Ni(II) complex, rather than the monomeric CAIH and its strong, square-planar Ni(II) complex, were found to be major catalysts of 8-oxo-2′-deoxyguanosine (8-oxo-dG) formation. The presence of Ni(II) largely enhanced 8-oxo-dG yield, especially at submillimolar concentrations of H2O2. The reaction was found not to involve detectable amounts of free radicals or Ni(III). These results, together with those published previously [Bal, W. et al. (1995) Chem. Res. Toxicol. 8, 683-692], lay a framework for the detailed investigations of the interactions of histone octamer with Ni(II) and other metal ions. They also suggest that molecular mechanisms of nickel carcinogenesis may involve oxidative damage processes catalyzed by weak Ni(II) complexes with cellular components.

Introduction Ni(II) compounds are established human carcinogens. The mechanism of their carcinogenic action is attributed in part to the enhancement of oxidative DNA base damage. Promutagenic 8-oxoguanine (8-oxo-Gua),1 being one of the major products of this process, may be involved in this mechanism (1). Reactivity of the Gua residue toward peroxides in the presence of Ni(II) complexes was studied in vitro in model systems containing 2′-deoxyguanosine (dG). The results generally support those of the in vivo studies (1-6). Therefore, dG oxidation is assumed to be a good indication of the carcinogenic potential of systems in question. Exposure of chromatin (but not pure DNA) to ambient oxygen in the presence of Ni(II) results in increased DNA base oxidation (7). Modulation of the damage by the protein component of chromatin clearly indicates complexation of Ni(II). Due to their abundance in chromatin, histones seem to be the most likely ligands. Among them, histone H3 is the only one containing an SH group which is capable of strong binding of Ni(II) at physiological pH. In our previous paper (8) we presented the

structural and thermodynamic description of the interaction of Ni(II) with CH3CO-Cys-Ala-Ile-His-NH2 (CAIH), a model peptide corresponding to the residues 110-113 of H3. The main complexes at physiological pH were Ni(CAIH)+ and Ni(CAIH)2 having a distorted square-planar geometry around Ni(II) and involving only His and Cys side chains in the binding. This binding mode is most likely involved in the interaction of Ni(II) with the whole protein. Formation of a Ni(II) complex with H3, which may be capable of oxygen activation, is likely to result in catalysis of DNA or free nucleotide oxidation. This promutagenic activity would be especially dangerous during cell replication, when vigorous de novo synthesis of both histones and DNA occurs, and a chance of direct interaction of Ni(II)-H3 complexes with nucleosides or nucleotides is very high. In this paper we present the results describing the enhancement of the dG oxidation with hydrogen peroxide by components of the CAIH-Ni(II) system. The purpose of this work was to use a well established model system to find out whether Ni(II) binding to the histone octamer may result in chemical processes that could lead to oxidative DNA damage.

Experimental Section * Address correspondence to this author at NCI-FCRDC, Building 538, Room 205, Frederick, MD 21702-1201. Tel: 301-846-1985; FAX: 301-846-5946. † National Cancer Institute. ‡ On leave from the Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland. § National Institute of Allergy and Infectious Diseases. X Abstract published in Advance ACS Abstracts, February 1, 1996. 1 Abbreviations: 8-oxo-Gua, 7,8-dihydro-8-oxoguanine; dG, 2′-deoxyguanosine; 8-oxo-dG, 7,8-dihydro-8-oxo-2′-deoxyguanosine; CAIH, CH3CO-Cys-Ala-Ile-His-NH2; HIAC-CAIH, [-S-(acetyl)-Cys-Ala-Ile-Hisamide]2; CAIH/Ni, solutions containing CAIH and Ni(II) ions; Ni(CAIH)+, a 1:1 complex of Ni(II) and CAIH; DMF, dimethylformamide; TFA, trifluoroacetic acid; DMPO, 5,5-dimethyl-1-pyrrolidine-N-oxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DSS, 3-(trimethylsilyl)propionic acid sodium salt; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy.

This article not subject to U.S. Copyright.

Materials.2 Hydrogen peroxide (H2O2), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), potassium ferricyanate [K3Fe(CN)6], and dG were purchased from Sigma Chemical Co. (St. Louis, MO). Nickel chloride (99.9999%), TFA, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Aldrich Chemical Co. 2 Certain commercial equipment and materials are identified in this paper in order to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Cancer Institute or the National Institute of Allergy and Infectious Diseases (National Institutes of Health, Department of Health and Human Services, U.S. Government) nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Published 1996 by the American Chemical Society

536 Chem. Res. Toxicol., Vol. 9, No. 2, 1996 (Milwaukee, WI). Chelex-100 chelating resin was purchased from Bio-Rad Laboratories (Richmond, CA) and monosodium and disodium phosphates were obtained from Fisher Scientific (Fair Lawn, NJ), methanol (HPLC grade) was purchased from EM Science (Gibbstown, NJ) and acetonitrile (HPLC grade) was obtained from J. T. Baker, (Phillipsburg, NJ). Reference sample of 8-oxo-dG was prepared by Dr. V. Nelson (SAIC-Frederick, Frederick, MD). The phosphate buffer and dG solutions were treated with Chelex-100 to remove possible transition metal contaminants. DMPO solution was purified using activated charcoal until free radical impurities disappeared as verified by ESR spectroscopy. Synthesis of Peptides. Acetyl-Cys-Ala-Ile-His-amide (CAIH). The peptide was synthesized according to the procedure described previously (8). [-S-(acetyl)-Cys-Ala-Ile-His-amide]2 (HIAC-CAIH). To a magnetically stirred solution of CAIH (7 mg, 14.5 µmol) in 30% aqueous acetonitrile was added 2 M aqueous ammonia to pH 8, followed by addition of 1 mM solution of K3Fe(CN)6 (60 mL, 60 µmol, 4 equiv). After stirring at 20 °C for 90 min, the progress of oxidation was checked by HPLC with detection at 215 nm, using ZORBAX 300 SB-C18, 300 Å, 5 µm, 250 × 4.6 mm column. The samples were eluted with a gradient system of 0.1% TFA/water (solvent A) and 0.1% TFA/80% acetonitrile/ water (solvent B): 100% A/0% B to 30% A/70% B over 40 min, flow rate 1 mL/min. Conversion of the starting material into a product was indicated by disappearance of a peak at 12.5 min and appearance of a new peak at 16.2 min. Composition of the product as CAIH dimer was confirmed by MALDI-TOF MS (m/z ) 964.9, calcd L + H ) 965.8). The dimer was purified on preparative HPLC on a Waters Delta-Pak (Milford, MA) C18 column, 300 Å pore size, 15 µm particle size, 19 × 300 mm, by elution with 0.1% TFA/water (solvent A) and 0.1% TFA/70% acetonitrile/water (solvent B), using a gradient of 95% A/5% B to 75% A/25% B over 40 min at a flow rate of 12.5 mL/min. Fractions containing product were pooled and lyophilized to yield 5 mg of fluffy, white solid of HPLC purity >98%. 8-Oxo-dG Formation. Solutions of 0.1 mM dG in 100 mM sodium phosphate buffer (pH 7.4) were incubated at 37 °C in the presence of 0 or 0.1 mM of CAIH, and/or Ni(II) and 0-100 mM H2O2. Incubation periods were 23 h in concentration dependency experiments and 10 min-48 h in time dependency experiments. After incubation, the dG samples were analyzed by HPLC without any additional pretreatment on a Waters Baseline 810 system equipped with a Waters 490E detector (detection at 254 nm) and an EG&G PAR Model 400 electrochemical detector (DC detection at 600 mV). A reversed-phase Beckman Ultrasphere C18 column (4.6 × 250 mm) was used. The mobile phase was 50 mM KH2PO4 solution in 12% aqueous methanol. Peptide Oxidation. Some of the samples used for 8-oxodG formation assay were also analyzed for peptide products on the same HPLC system as 8-oxo-dG analysis, using the 0.1% TFA/water (solvent A) and 0.1% TFA/80% acetonitrile/water (solvent B), gradient system of 100% A/0% B to 30% A/70% B over 40 min, flow rate 1 mL/min, and detection at 215 nm. Cyclic Voltammetry. Measurements were performed at 25 °C with a Bioanalytical Systems (BAS) Model 100B Electrochemical analyzer using BAS d ) 3 mm platinum and glassy carbon electrodes with solutions containing 1 mM CAIH and 0-1 mM Ni(II) in 100 mM sodium phosphate buffer (pH 7.4). ESR. Attempts to detect formation of Ni(III) complexes were performed with a Bruker ESP-300 spectrometer operating at 9.4 GHz with a 100 KHz modulation and DPPH as standard. Samples containing 0.1-1 mM CAIH and Ni(II) and 10 mM H2O2 were frozen in liquid N2 after 5-20 min incubations at room temperature and measured at 120 K. Spin-trapping experiments with DMPO were done at room temperature with a Varian E3 spectrometer operating at 9.4 GHz with a 100 KHz modulation frequency and using DPPH as a standard. Sample concentrations were as follows: 1mM CAIH, 1 mM Ni(II), 10 mM H2O2, 1 M DMPO, and 100 mM phosphate buffer

Bal et al. (pH 7.4). Experimental procedures were similar to those used previously (9). UV/Vis. Spectra were recorded at 25 °C on a Beckman DU640 spectrophotometer over the spectral range of 190-1100 nm with samples containing 0-1 mM Ni(II) and/or 0.1-1 mM CAIH, or 0-0.03 mM HIAC-CAIH, using 1 or 5 cm cuvettes. CD. Spectra were recorded at 25 °C on a Jasco J-500A spectropolarimeter equipped with an IF-500 analog-to-digital interface and controlled by Jasco software operating on an IBMPC compatible. The spectra were recorded over the range of 195-400 nm, using 1 cm cuvettes. Peptide concentrations were 1.0 and 0.1 mM. Concentration of HIAC-CAIH of 10 µM and peptide-to-metal ratios between 1:0 and 1:100 were used. Spectra are expressed in terms of ∆ ) l - r. Potentiometry. The protonation and stability constants of binary and ternary Ni(II) complexes of phosphate ions and CAIH in the presence of 0.1 M KNO3 were determined at 25 °C using pH-metric titrations over the pH range 3-11.5 (Molspin automatic titrator, Molspin Ltd., Newcastle-upon-Tyne, U.K.) with NaOH as titrant. Changes of pH were monitored with a combined glass-calomel electrode calibrated daily in hydrogen concentrations by HNO3 titrations (10). Sample volumes of 1.5 -2.0 mL and concentrations of 1 mM CAIH, 0.5-1 mM NiCl2, and 1-3 mM monosodium phosphate were used. The data were analyzed using the SUPERQUAD program (11). Standard deviations computed by SUPERQUAD refer to random errors.

Results Phosphate Effect on Ni(II) Complexation with CAIH. All reactivity studies toward dG were performed in 100 mM phosphate buffer. Therefore, its influence on coordination equilibria between CAIH and Ni(II) had to be considered. This was achieved by two approaches. First, CAIH/Ni solutions at pH 7.4 were titrated with phosphate buffer of the same pH and monitored by UV/ vis spectroscopy (A335). Extent of vanishing of the Ni(CAIH)+ spectral pattern at low phosphate concentrations (see Figure 1) was larger than predicted for the sole formation of competing binary nickel-phosphate complexes, and therefore indicated the formation of a ternary complex. Also, in the visible range of the spectra, vanishing of the spectrum of the parent square-planar complex Ni(CAIH)+ and emergence of a new transition at 600 nm ( ∼ 7) characteristic for octahedral complexes were seen (see inset in Figure 1A). Second, pH-metric titrations of parent binary systems and of the potential ternary system were performed. Stability constants calculated from titrations of CAIH and CAIH/Ni systems were reproducible to within experimental error compared to those previously published (8). Titrations of solutions containing CAIH, Ni2+, and phosphate also gave evidence for the formation of a ternary complex. Calculations yielded Ni(CAIH)(H2PO4) as the only ternary species detectable. Complex of this stoichiometry also gave the best fit to spectrophotometric titrations. Stability constants obtained in this work are presented in Table 1. For comparison, available literature data (12, 13) are also presented. Speciation diagram calculated for typical concentrations used in the 8-oxo-dG formation experiments on the basis of potentiometric results is presented in Figure 2. Nickel Binding by HIAC-CAIH. Solubility of HIAC-CAIH in phosphate buffer is very limited. It was measured by a titration of 5 mL samples of 100 mM phosphate buffer (pH 7.4) with 1 mM solution of HIACCAIH in methanol. UV spectra were recorded at each of forty 1-µL additions of the latter. Light scattering due to the presence of a precipitate was detected at 0.031

Effect of Ni(II)-CAIH Complexes on dG Oxidation

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 537 Table 1. Protonation and Stability Constants of Ni(II)-CAIH-Phosphate System at I ) 0.1 M and 25 °Ca species

log βb

H(CAIH)H2(CAIH) Ni(CAIH)+ Ni(CAIH)2 NiH-1(CAIH) NiH-2(CAIH)NiH-3(CAIH)2HPO42H2PO4H3PO4 NiHPO4 NiH2PO4+

8.58(1) 15.03(1) 4.04(6) 8.02(5) -4.90(8) -13.92(9) -22.88(4) 11.39(2) 18.19(2) 20.26(5) 13.4(2) undetected

Ni(CAIH)(H2PO4)f

26.31(6)e, 25.78(3)f

log βc,d 8.59 15.04 4.03 7.98 -4.89 -13.87 -22.85 11.54 18.13 20.13 13.66 18.63

KT ) [Ni(CAIH)H2PO4]/[H2PO4-][Ni(CAIH)+] log KT ) 4.08e, 3.55f a β(Ni H L A ) ) [Ni H L A ]/[Ni]i[H]j[L]k[A]l. Standard deviai j k l i j k l tions on the last digit are given in parentheses. b This work. c Ref 8 for CAIH. d Phosphate constants calculated from data in refs 12 and 13. e Potentiometry. f Spectrophotometry.

Figure 1. (A) Spectra of a solution of 0.5 mM NiCl2 and 0.5 mM CAIH in the presence of sodium phosphate buffer (pH 7.4) at the following concentrations: 1, 0; 2, 5; 3, 12; 4, 18; 5, 27; 6, 36; 7, 70; and 8, 115 mM. The inset shows spectral bands characteristic for octahedral Ni(II) complexes at 18-115 mM phosphate: a, Ni(CAIH)(H2PO4); b, NiHPO4. (B) Effect of addition of 100 mM phosphate on the absorption spectrum of a sample containing 1 mM NiCl2 and 1 mM CAIH at pH 7.4: (s) without phosphate, (‚‚‚) with phosphate.

mM HIAC-CAIH and at 1% methanol. Therefore, it was assumed that HIAC-CAIH solutions are saturated at 0.03 mM under the conditions of our assays. In order to observe Ni(II) binding to HIAC-CAIH and a possible effect of phosphate thereon, CD spectra of the peptide at various pH were recorded. Then the effect of Ni(II) on the far-UV CD pattern with and without phosphate at pH 7.4 was examined. The CD spectra, presented in Figure 3, clearly indicate a specific influence of Ni(II) in the far UV at high Ni(II):HIAC-CAIH ratios. This effect is partially canceled by phosphate ions. Formation of 8-Oxo-dG. Yield of 8-oxo-dG generated with CAIH-Ni(II) and HIAC-CAIH-Ni(II) systems as function of H2O2 concentration is presented in Figure 4. Two modes of sample preparation were adopted in CAIH/Ni(II) experiments, differing by the order and timing of additions of components. In mode A, hydrogen peroxide was added last after allowing samples to equilibrate for 5 min at 20 °C. This time was estimated on the basis of observations presented in (8) to be fully sufficient to achieve coordination equilibrium. In mode

Figure 2. Species distribution diagram for concentrations used in dG oxidation experiments: 0.1 mM Ni2+, 0.1 mM CAIH, 100 mM phosphate.

Figure 3. Effect of pH, phosphate, and Ni(II) on the CD spectra of HIAC-CAIH. (a) Effect of pH: (‚‚‚) pH 3.0, (s) pH 7.4, (- - -) pH 11.0. (b) Effect of Ni(II) at pH 7.4: (- - -) 1 equiv of Ni(II), (‚‚‚) 10 equiv of Ni(II), (s) 100 equiv of Ni(II). (c) Effect of Ni(II) at pH 7.4 and 100 mM phosphate: (- - -) 1 equiv of Ni(II), (‚‚‚) 10 equiv of Ni(II), (s) 100 equiv of Ni(II).

B, dG was added last, 1 h after incubating all other components at 20 °C. This procedure was aimed at

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Bal et al.

Figure 4. Dependence of the 8-oxo-dG yield from 0.1 mM dG on hydrogen peroxide concentration. Initial concentrations of other reagents as in Figure 2.

Figure 6. Oxidation of CAIH in the course of dG-H2O2 reaction in the absence (A) and presence (B) of Ni(II). 1, 2: major further products of CAIH oxidation beyond disulfide. Initial concentrations of reagents as in Figures 2 and 5B.

samples without H2O2 the yield of 8-oxo-dG was at the background level of 0.1 ( 0.05 µg/mg of dG, regardless of their composition, and was not statistically different from control samples containing only dG (data not shown). Fate of the Peptide in Reaction Mixture. Chromatograms of the peptide products formed in the course of reaction of dG with 1 mM H2O2 in the absence and presence of Ni(II) are shown in Figure 6. Main products were identified by comparison with reference chromatograms. Figure 5. Dependence of the 8-oxo-dG yield on incubation time at (A) 10 mM H2O2 or (B) 1 mM H2O2. Initial concentrations of other reagents as in Figures 2 and 4.

examining the potential involvement of substrates and/ or early products of the H2O2 reaction with CAIH and Ni(CAIH)+ in dG oxidation. Samples were inspected for the presence of precipitate prior to injection into HPLC, and such was never observed. Figure 5 presents the 8-oxo-dG yield as a function of time for 1 and 10 mM H2O2 (mode A). Change of dG concentration was within the experimental error of HPLC analysis ((2%). In all

Formation of Ni(III) or Radical Species. No evidence for the involvement of radicals, which could be trapped by DMPO, or for Ni(III) formation in the mixtures with dG was obtained. Reactions with oxygen or hydrogen peroxide monitored with ESR did not produce any detectable signals both at room temperature and after freezing to liquid N2 temperature. Attempts to obtain redox potentials for the Ni(CAIH)+ complex in the -0.5 to +1.0 V range in the absence or presence of H2O2 also failed to indicate any reversible electrode processes in these systems.

Effect of Ni(II)-CAIH Complexes on dG Oxidation

Discussion Interaction of CAIH Peptides with Ni(II) and Phosphate. Binding of H2PO4- to Ni(CAIH)+ induces a complex rearrangement from square-planar to octahedral. This is evidenced by vanishing of square-planar ligand field bands of Ni(II) and corresponding charge transfer bands and by appearance of new bands typical for octahedral complexes (see Figure 1). This is a phenomenon often encountered in Ni(II) complexes when a slight change in the in-plane ligand field, availability of an axial ligand, or even sterical interactions between ligand molecules can shift the equilibrium completely (14). All these mechanisms are possible in this case. This effect additionally supports the conclusion in our previous paper (8) about the distorted, and geometrically labile, character of the Ni(CAIH)+ complex. An underestimation of stability of the octahedral ternary species in spectrophotometric experiments by ∼0.5 log unit (see Table 1) may be due to its optical isomerism, resulting in a minor square-planar complex of the same stoichiometry. Such hypothetical species can be expected to absorb at or near 335 nm and therefore systematically increase the calculated concentration of the unreacted parent squareplanar complexes, resulting in the underestimation of the equilibrium concentration of the octahedral Ni(CAIH)(H2PO4) complex. The formation constant of the ternary complex is very high; e.g., log KT value is more than 1 order of magnitude higher than that of the ternary Ni(II)-ATP-imidazole complex, 2.44 (15). This indicates a strong attraction between coordinated ligands which may be of importance for Ni(II) interaction with histone H3 in physiological medium. The disulfide-bridged dimer of CAIH, HIAC-CAIH, is not capable of binding nickel ions through sulfur. This leaves only imidazole ring as a potential binding site. Involvement of amide nitrogens is not possible at pH 7.4, and additionally, such a process would lead to the formation of a square-planar complex (8, 16). This is obviously not the case. An octahedral imidazole-bound complex could not be detected by electronic spectroscopy. The value of  even for the most intense d-d band at around 385 nm cannot be expected to exceed 15 (17). The detection limit in our experimental conditions was 0.001 AU, which therefore corresponds to the complex concentration of at least 1.5 × 10-5 M. (Ni(II)-phosphate complexes with 385 values around 6 were not detected under these conditions.) Obviously, stability of HIACCAIH-Ni(II) cannot be nearly as high as that of the Ni(CAIH)+ complex and should rather be comparable to that of Ni(II)-imidazole (18). Calculations for a hypothetical system including imidazole and phosphate suggest that in 100 mM phosphate (pH 7.4) total concentration of Ni(II)-imidazole complexes is below 0.2% of total nickel. The equilibrium concentration of HIAC-CAIHNi(II) under our experimental conditions can therefore be expected to be of the range of 6 × 10-8 M, thus 250 times lower than the detection limit for ligand field bands. Positive, although only qualitative, evidence for weak binding could, however, be seen in the far-UV CD spectra. A new band appeared at 217 nm upon addition of 10-fold and especially 100-fold excess of Ni(II) over HIAC-CAIH. Position of this band is perfectly coincident with that reported previously (19) for Ni(II) complexes of histidine and His-containing peptides. This band is most likely a charge transfer between the

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 539

imidazole nitrogen and octahedral Ni(II), but regardless of its exact nature it can serve as a marker of Ni(II) coordination. The presence of 100 mM phosphate largely suppressed this band, and therefore, presumably, the Ni(II) binding to the peptide. Unfortunately, addition of further Ni(II) equivalents resulted in precipitation of nickel phosphate and the band could not be investigated in more detail. Generation of 8-Oxo-dG. Nickel and CAIH alone are capable of facilitating hydroxylation of dG by hydrogen peroxide. This activity is, however, enhanced in systems containing both components, especially at low, submillimolar H2O2 concentrations and shorter incubation times (Figures 4 and 5). The enhancement does not involve any detectable radical and/or Ni(III) intermediates. Control experiments with the use of synthetic dimer and preoxidized monomer (mode B on Figure 4) revealed that the very weak species containing Ni(II), HIAC-CAIH, and presumably phosphate is the only one correlating well with the 8-oxo-dG enhancement. There may be some involvement of the Ni(CAIH)+ complex at H2O2 concentrations below 2 mM, but the differences of yields between mode A, mode B, and HIAC-CAIH reactions are on the edge of statistical significance in that range. Binding of dG to Ni(II) is so weak (3) that it can safely be neglected for experimental conditions applied. The lower 8-oxo-dG yields with synthetic HIAC-CAIH, as compared to those with CAIH, are roughly proportional to the difference between its solubility and maximal theoretical concentration (0.03 and 0.05 mM, respectively). They therefore seem to indicate formation of oversaturated solutions of HIAC-CAIH in the case of in situ oxidation of CAIH, especially as no precipitate was found in reaction mixtures. These findings are further supported by the observation that HIAC-CAIH is the main product of H2O2 oxidation of CAIH in both the absence and presence of Ni(II) in our experimental conditions. Formation of further peptidic products does not correlate with the time course of 8-oxo-dG formation. If any of the new products were a major catalyst then a surge of 8-oxo-dG formation would be expected after several hours. The opposite is true (Figure 5). The free thiol of the CAIH molecule plays a surprisingly little role in the oxidation process. Usually, the catalysis of oxidative processes in the presence of thiols is attributed to the radical-driven oxidation of thiols (20). Not only the radicals were missing in our reaction mixtures, but their content of the thiol was negligible already after 2 h of incubation at 37 °C (Figure 6). At that and longer times, the 8-oxo-dG formation is very effective (Figure 5). Formation of the Ni(II)/Ni(III) redox couple and related generation of radicals is a characteristic feature of complexes containing square-planar Ni(II), e.g., Nitetraglycine (5), or Ni-Gly-Gly-His (21). Such systems easily oxidize dG beyond the 8-oxo-dG step and also induce other kinds of damage, like depurination. The octahedral ones, e.g., Ni(II)-histidine (22), can also catalyze dG hydroxylation, but seem to be more specific for 8-oxo-dG. In the CAIH system, yields of 8-oxo-dG were comparable to or higher than those achieved with histidine (22), histidine-containing peptides (4), or even tetraglycine (5), despite the fact that concentrations of reagents applied in this work were 1 order of magnitude lower. Unlike with tetraglycine, no loss of dG, except for transformation into 8-oxo-dG, was observed. All the evidence presented above is consistent with the minor, weak octahedral Ni(II) complex with HIAC-CAIH

540 Chem. Res. Toxicol., Vol. 9, No. 2, 1996

as the catalyst and indicates its extremely high specific activity toward dG hydroxylation.

Bal et al.

(6)

Conclusion This study resulted in two unexpected findings. First, the thiolate and thiolate-bound nickel seem to be only marginally involved in the catalysis of dG oxidation, at least in the CAIH system. Second, amounts of 8-oxo-dG formed in the presence of both the relatively redox-inert disulfide-bridged HIAC-CAIH, and its apparently loosely bound and nonspecific octahedral nickel complex, were extremely high given the presumably very low solution concentration of the active species. These results thus seem to indicate a possibility that weak and thus often disregarded nickel complexes may have a very high mutagenic potential. The complex with oxidized glutathione and other cellular components may therefore be very important in mutagenic processes in the cell. The involvement of histone H3 in DNA or nucleotide oxidation during cell replication, when these components are synthesized de novo and are therefore abundant in the free rather than assembled form, is also possible in light of the results presented above. The other result, the detection of a ternary complex involving Ni(II), CAIH, and phosphate, is very important for the study of Ni(II) binding and reactivity toward the histone octamer, currently under way in our laboratory.

(7)

(8)

(9)

(10)

(11)

(12) (13)

(14) (15)

Acknowledgment. The authors wish to thank Dr. Malgorzata Jezowska-Bojczuk for help with potentiometric measurements and calculations, Dr. Keith M. Davies for assistance with CV measurements, Drs. Xianglin Shi and Marc Desrosiers for help with ESR experiments, Dr. Joseph E. Saavedra and Dr. Michael P. Waalkes for helpful comments on the manuscript, and Dr. Anthony Dipple for making his CD spectrometer available to us.

References (1) Kasprzak, K. S. (1995) Possible role of oxidative damage in metalinduced carcinogenesis. Cancer Invest. 13, 411-430. (2) Kasprzak, K. S., and Hernandez, L. (1989) Enhancement of hydroxylation and deglycosylation of 2′-deoxyguanosine by carcinogenic nickel compounds. Cancer Res. 49, 5964-5968. (3) Datta, A. K., Riggs, C. W., Fivash, M. J., Jr., and Kasprzak, K. S. (1991) Mechanisms of nickel carcinogenesis. Interaction of Ni(II) with 2′-deoxynucleosides and 2′-deoxynucleotides. Chem.-Biol. Interact. 79, 323-334. (4) Datta, A. K., Shi, X., and Kasprzak, K. S. (1993) Effect of carnosine, homocarnosine and anserine on hydroxylation of the guanine moiety in 2′-deoxyguanosine, DNA and nucleohistone with hydrogen peroxide in the presence of nickel(II). Carcinogenesis 14, 417-422. (5) Datta, A. K., North, S. L., and Kasprzak, K. S. (1994) Effect of nickel(II) and tetraglycine on hydroxylation of the guanine moiety

(16)

(17) (18) (19) (20) (21)

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