Generation of Putative Intrastrand Cross-Links and Strand Breaks in

Chem. Res. Toxicol. 1997, 10, 393-400

393

Generation of Putative Intrastrand Cross-Links and Strand Breaks in DNA by Transition Metal Ion-Mediated Oxygen Radical Attack Daniel R. Lloyd,* David H. Phillips, and Paul L. Carmichael† Section of Molecular Carcinogenesis, Haddow Laboratories, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Received September 9, 1996X

Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 108 nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 µM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.

Introduction It is well established that free radicals are formed in living organisms as a function of endogenous biochemical processes. Of particular relevance in aerobic systems are those reactive species derived from the metabolism of oxygen, which include hydrogen peroxide, singlet oxygen, superoxide radical, and the hydroxyl radical, collectively known as reactive oxygen species (ROS).1 Significant intracellular concentrations of ROS are thought to be produced during normal aerobic metabolism (1). Overproduction of ROS in cells has been demonstrated to result from exposure to environmental pollutants (2) and ionizing radiation (3), while sites of tissue inflammation are known to have enhanced levels of ROS (4). Indeed, elevated levels of ROS in cells have been implicated in the aetiology of a number of pathologies, including * To whom correspondence and requests for reprints should be addressed at the Haddow Laboratories, Institute of Cancer Research, Cotswold Rd., Sutton, Surrey SM2 5NG, U.K. Fax: 0181 770 7290. E-mail: [email protected]. † Present address: Imperial College School of Medicine at St. Mary’s, Department of Pharmacology and Toxicology, Norfolk Place, London W2 1PG, U.K. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1 Abbreviations: ROS, reactive oxygen species; 8-OHdG, 8-hydroxydeoxyguanosine; PEI, poly(ethylenimine); dApdA, 2′-deoxyadenylyl(3′-5′)-2′-deoxyadenosine; dApdG, 2′-deoxyadenylyl(3′-5′)-2′-deoxyguanosine; B[a]P, benzo[a]pyrene; B(g)C, benzo[g]chrysene; PAH, polycyclic aromatic hydrocarbons.

S0893-228x(96)00158-0 CCC: $14.00

arthritis (5), cataracts (6), multiple sclerosis (7), ageing (8), and cancer (9). Intracellular formation of ROS, such as the hydroxyl radical, can bring about extensive damage to all biological macromolecules within the cell, including protein fragmentation (10), lipid peroxidation (11), carbohydrate degradation (12), and damage to genetic material. DNA, with its numerous negatively charged sites, is a particularly good target for the electrophilic hydroxyl radical (13). Damage to DNA following hydroxyl radical attack includes the production of single base modifications, such as 8-hydroxydeoxyguanosine (8-OHdG) (14), thymidine glycols (15), and DNA strand breaks (16-18). Subsequent replication of oxidatively-damaged DNA can result in double or “tandem” mutations (19, 20) and single base substitutions (21). There is growing evidence that transition metals are able to interact with and alter genetic material via an oxidative mechanism involving the production of free radicals (11). The complex redox chemistry of transition metal ions allows them to catalyze a number of oneelectron oxidations/reductions, including the Fenton-type reduction of hydrogen peroxide to form the hydroxyl radical (22). For example, nickel and chromium, both recognized as human carcinogens, produce 8-OHdG and strand breaks when their salts are incubated with DNA in vitro in the presence of cellular chemicals such as ascorbate, glutathione, and hydrogen peroxide (23, 24). © 1997 American Chemical Society

394 Chem. Res. Toxicol., Vol. 10, No. 4, 1997

Copper and iron salts are also known to cause strand breaks when incubated with DNA and hydrogen peroxide in Fenton-type systems (25), and there is evidence that cobalt (26) and vanadium (27) exhibit similar reactivity. It has been shown that exposure of DNA to a Fentontype reaction involving copper(II) sulfate or iron(II) sulfate also results in the production of DNA lesions, detectable by 32P-postlabeling, that have similar chromatographic properties to bulky carcinogen-DNA adducts (28, 29). These lesions have been proposed to be formed by an intrastrand reaction resulting in the dimerization of adjacent purine bases in DNA; in particular, two major products of this type were identified as dimers of adenine interacting with adenine, and of adenine interacting with guanine, respectively (29). Similar structures have been proposed recently from experiments using oligonucleotides of defined sequence (30). In the present study, we demonstrate, using the 32 P-postlabeling assay, the production of bulky/hydrophobic lesions in DNA treated with a number of other transition metals in Fenton-type radical-generating systems and compare the formation of these putative intrastrand cross-links with concomitant strand break formation.

Materials and Methods Materials. Salmon sperm DNA, 2′-deoxyadenylyl(3′-5′)-2′deoxyadenosine (dApdA), 2′-deoxyadenylyl(3′-5′)-2′-deoxyguanosine (dApdG), ethidium bromide, cobalt(II) sulfate, cadmium(II) chloride, bovine catalase (C9284), and iminodiacetic acid (I4758) (both attached to beaded agarose) were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Vanadium(III) chloride was obtained from Aldrich (Gillingham, Dorset, U.K.). Copper(II) sulfate, zinc(II) sulfate, and hydrogen peroxide were obtained from BDH (Poole, Dorset, U.K.). Iron(II) sulfate and EDTA were obtained from Fisons (Loughborough, Leicestershire, U.K.). Nickel(II) sulfate, sodium chromate(VI), and chromic(III) chloride were obtained from Hopkin and Williams (Chadwell Heath, Essex, U.K.). Low melting point agarose (electrophoresis grade) and a 1 kb DNA molecular weight ladder were obtained from Life Technologies (Paisley, Renfrewshire, Scotland). Chemicals and materials used in the 32P-postlabeling assay were obtained from previously mentioned sources (31). Oxygen Radical Treatment of DNA. Salmon sperm DNA was repurified using the phenol extraction method (32), and dissolved in deionized water. For some experiments, it was further purified by dialysis against progressively weaker solutions of EDTA, and finally against distilled water. Aliquots (500 µL, 0.4 mg/mL) were incubated for 1 h at 37 °C with 50 mM H2O2 and between 25 µM and 1 mM of copper(II) sulfate, iron(II) sulfate, nickel(II) sulfate, cobalt(II) sulfate, cadmium(II) chloride, vanadium(III) chloride, chromic(III) chloride, sodium chromate(VI), or zinc(II) chloride. The reaction was stopped by adding immobilized catalase (100 µL). The insoluble enzyme was removed after 15 min by centrifugation (using an Eppendorf Microfuge, Model 5415) at 15 000 rpm for 5 min. DNA in the supernatant was precipitated by adding sodium chloride (5 M, 0.1 volume) followed by ice-cold ethanol (2 volumes) and then leaving overnight at -20 °C. When precipitation of treated DNA was not possible, due to extensive fragmentation, immobilized iminodiacetic acid (100 µL) was added to the supernatant in order to remove the transition metal ions by chelation. After 15 min, the suspension was centrifuged as before and the supernatant stored at -20 °C prior to 32P-postlabeling. 32P-Postlabeling. DNA samples were subjected to the nuclease P1 enrichment method of postlabeling analysis (33). Aliquots containing 4 µg of DNA were taken from the thawed samples and evaporated to dryness using a Savant SpeedVac

Lloyd et al. SVC100 vacuum centrifuge. The DNA was digested overnight at 37 °C by the addition of micrococcal nuclease (0.14 unit) and spleen phosphodiesterase (1.2 µg) in 20 mM sodium succinate/ 10 mM calcium chloride (pH 6.0, 4.8 µL). Samples were then further digested for 1 h at 37 °C with nuclease P1 (0.15 unit). 32P-Labeling of each sample was carried out by incubation at 37 °C for 30 min with [γ-32P]ATP (50 µCi, specific activity approximately 4000 Ci/mmol) and T4 polynucleotide kinase (6 units). Chromatographic Resolution of DNA Lesions. After radiolabeled digests were applied to the origins of 10 × 10 cm PEI-cellulose TLC plates, multidirectional chromatography was carried out using the following solvent systems: D1, 1.0 M sodium phosphate (pH 6.0) onto a paper wick; D2, 3.5 M lithium formate, 8.5 M urea (pH 3.5); D3, 0.8 M lithium chloride, 0.5 M Tris-HCl, 8.5 M urea (pH 8.0); D4, 1.7 M sodium phosphate (pH 6.0) onto a paper wick. In some experiments, 2.3 M sodium phosphate (pH 5.8) was used as the D1 solvent. DNA lesions were detected as radioactive spots on the TLC plates after screen-enhanced autoradiography at -80 °C for 24 h using Kodak scientific imaging film (Eastman Kodak, Rochester, NY, product no. 165 1454). Quantification of these lesions was carried out using an autograph two-dimensional radioisotope imaging system (Oxford Positron Systems, Oxford, U.K.), which responds linearly to cpm. HPLC Analysis of Radiolabeled DNA Lesions. HPLC analysis was carried out using the method of Pfau and Phillips (34). The apparatus consisted of two Waters 501 HPLC pumps, a Waters 712 WISP autosampler, a Waters 440 absorbance detector, and a Berthold LB 507 HPLC radioactivity monitor. Waters Baseline-810 software was used to achieve gradient control and data processing. Radioactive spots were excised from the PEI-cellulose sheets; the radioactive material was then eluted by shaking overnight in pyridinium formate (500 µL, 4 M, pH 4.5). After filtering, the sample was evaporated to dryness, redissolved in water (50-200 µL), and then injected onto a Zorbax phenyl-ODS reverse phase column (Hichrom, Reading, U.K.). A phosphate buffer/methanol gradient was employed as described previously (29). Agarose Gel Electrophoresis. Aliquots (2 µg) of the same Fenton-treated oxidized DNA samples were loaded onto 2% agarose gels (100 mL) containing ethidium bromide (0.5 µg/mL) and TAE electrophoresis buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) (35). Electrophoresis was for 3 h at 60 V, after which the gels were viewed under UV light.

Results Fenton-Type Reactions with DNA. Treatment of salmon sperm DNA with copper(II) sulfate and hydrogen peroxide at a range of concentrations was found to result in the production of eight bulky DNA lesions resolved by two-dimensional thin-layer chromatography (Figure 1A, spots 1-8). Spots 1 and 2, which accounted for 14.7% and 42.4% of the total products detected at 250 µM, were the major products in the copper(II) Fenton system, confirming results seen previously (29). Further investigations substituting sodium chromate(VI), cobalt(II) sulfate, iron(II) sulfate, nickel(II) sulfate, or vanadium(III) chloride in place of copper(II) sulfate resulted in the detection, by postlabeling, of between four and six discrete products (Figure 1B-F, spots 1-9). Spots 1 and 2 were common to each of these metal ion/hydrogen peroxide systems. In addition to these two products, three other products (spots 3, 4, and 5) were consistently produced in the chromate(VI), iron(II), and nickel(II) Fenton systems (Figure 1B,D,E). Similarly, spots 3, 4, and 5 were produced in the cobalt(II) Fenton system (Figure 1C), as well as an additional one (spot 7). The vanadium(III) Fenton system generated spots 3, 4, and

Oxidative DNA Damage

Figure 1. Autoradiograms of PEI-cellulose TLC separations of 32P-postlabeled samples of DNA or dinucleotides treated with ROS. Treatment was with Fenton-type radical-generating systems involving hydrogen peroxide and transition metal compounds as follows: (A) copper(II) sulfate; (B) sodium chromate(VI); (C) cobalt(II) sulfate; (D) iron(II) sulfate; (E) nickel(II) sulfate; (F) vanadium(III) chloride; (G) H2O2 only; (H) cadmium(II) chloride; (I) chromic(III) chloride; (J) zinc(II) chloride; (K) dApdG, copper(II) sulfate, H2O2; (L) dApdA, copper(II) sulfate, H2O2. Autoradiography was at -80 °C for 3 days for samples G-J; 24 h for all other samples. (Exposure for less than 24 h demonstrates more clearly the presence of the two discrete major products; however, the minor products are less clearly visible. Hence, in order to demonstrate the presence of these minor products, autoradiography for 24 h is necessary, and consequently some panels, notably A and C, appear overexposed.)

5 inconsistently and at levels much lower than in the other Fenton systems. Spot 9, however, was generated and was unique to this system (Figure 1F). In similar experiments in which the more concentrated D1 solvent was used (2.3 M sodium phosphate instead of 1 M), no additional TLC spots were observed (data not shown). No bulky DNA lesions were detected in the absence of hydrogen peroxide. Incubation of DNA with hydrogen peroxide alone resulted in the formation of very low levels of DNA lesions (Figure 1G). These spots of radioactivity were barely visible when the incubation involved DNA that had been extensively dialyzed, suggesting that the lesions were due to contamination of the commercially obtained DNA with trace metal ions. Zinc(II), chromium(III), and cadmium(II) did not generate levels of bulky lesions, when incubated with DNA and hydrogen peroxide, that were significantly different from those obtained with DNA and hydrogen peroxide only (Figure 1H-J). (Note that chromatograms 1G-J were subjected to autoradiography for 3 times as long as the other chromatograms in the figure.)

Chem. Res. Toxicol., Vol. 10, No. 4, 1997 395

Formation of all the bulky DNA lesions was found to depend on the concentration of the transition metal ion used in each Fenton system. Following an initial increase in formation of all products in the copper(II) Fenton system, the levels declined above a concentration of 250 µM copper(II) sulfate and approached zero at 1 mM. In contrast, increasing the concentration of chromate(VI) from 25 µM to 1 mM resulted in a steady increase in the yield of all five products (Figure 2B). Similarly, in the cobalt(II) Fenton system, an increase in metal ion concentration from 25 mM to 1 mM resulted in an increase in yield of the same products except spot 1, whose yield decreased at metal ion concentrations above 600 µM (Figure 2C). The production of DNA lesions in the iron(II) Fenton system reached a maximum at 150 µM before declining and reaching zero at concentrations above 250 µM (Figure 2D). Increasing the concentration of nickel(II) sulfate from 25 µM to 1 mM resulted in a steady increase in the yield of all five DNA lesions generated in this Fenton system (Figure 2E). Increasing the concentration of vanadium(III) chloride above 150 µM resulted in a decline in the yield of DNA lesions and reached zero at concentrations above 400 µM (Figure 2F). Table 1 shows the abundance of each bulky product generated in all the Fenton systems relative to the total yield of products. These data were taken at the metal concentration where the total yield of bulky lesions was highest. Spots 3 and 4, which were produced in comparable levels in five of the Fenton systems, both amounted to between 8 and 13% of total formation of bulky lesions in the cobalt(II) and copper(II) systems, while spot 2 accounted for between 45 and 49%. However, in the chromium(VI), nickel(II), and iron(II) systems, the levels of products 3 and 4 amounted to a greater percentage (up to 22.2%) of total formation of these lesions. Indeed, the difference in production of products 3 and 4 between these Fenton systems was all the more significant considering the reduction in formation of major product 2 (as low as 27.0% total bulky lesion formation). Spot 5 accounted for a greater proportion of total DNA lesions (13.2%) in the iron(II) Fenton system than in the other Fenton systems (3.0-6.8%). Spot 7 was present only in the copper(II) and cobalt(II) Fenton systems, while spot 6 [from the copper(II) Fenton system] and spot 9 [from the vanadium(III) Fenton system] were the only products that were unique to one Fenton system. An experiment was carried out in order to determine whether residual transition metal ions in the DNA samples affected the efficiency of the postlabeling assay. Each of the nine transition metal ions were added to aliquots of benzo[a]pyrene- and benzo[g]chrysene-modified DNA to a concentration of 1 mM, and the DNA was then purified by either ethanol precipitation or treatment with immobilized iminodiacetic acid, depending on which method had been used in the corresponding Fenton incubations. The efficiency of total adduct labeling, compared to labeling of B[a]P- and B[g]C-modified DNA that had not been treated with transition metal ions, was found to range from 45 to 82%. The exception was chromium(III), which almost completely inhibited postlabeling of PAH-DNA adducts. HPLC Analyses. In order to confirm the same identity of the major bulky lesions produced in DNA by each of the metal ion/hydrogen peroxide radical-generating systems, reverse phase HPLC was carried out. Spot 1, generated in each Fenton system (Figure 1A-F), gave


Lloyd et al.

Figure 2. Effect of transition metal ion concentation on the mean levels of bulky DNA lesions produced in the Fenton-type radicalgenerating systems. Salmon sperm DNA was exposed to 50 mM hydrogen peroxide and between 25 and 1000 µM transition metal ion as follows: (A) copper(II) sulfate; (B) sodium chromate(VI); (C) cobalt(II) sulfate; (D) iron(II) sulfate; (E) nickel(II) sulfate; and (F) vanadium(III) chloride. Error bars represent the SEM for duplicate determinations of duplicate samples. Table 1. Relative Abundance in DNA of Bulky Lesions, Detected by 32P-Postlabeling, Induced by Transition Metal Ions and Hydrogen Peroxidea percentage of total radioactivity

spot no.

copper(II) sulfate (250 µM)

sodium chromate(VI) (1 mM)

cobalt(II) sulfate (800 µM)

iron(II) sulfate (150 µM)

nickel(II) sulfate (1 mM)

vanadium(III) chloride (150 µM)

1 2 3 4 5 6 7 8 9

14.7 42.4 12.6 10.3 5.4 4.1 7.6 3.0 n/d

27.0 27.0 17.1 22.2 6.8 n/d n/d n/d n/d

27.6 48.5 12.6 8.8 3.0 n/d 10.4 n/d n/d

24.2 31.2 13.0 18.4 13.2 n/d n/d n/d n/d

26.8 41.6 13.6 14.4 3.5 n/d n/d n/d n/d

37.3 41.8 n/db n/d n/d n/d n/d 5.7 15.2

a For each transition metal compound, the value shown in parentheses is the transition metal ion concentration at which total formation of bulky lesions was the highest. b n/d: not detected.

very similar HPLC chromatograms and a mean retention time of 18.7 ( 0.5 min. Figure 3A shows an HPLC chromatogram of spot 1 generated in the copper(II) Fenton system. Similar analysis of spot 2 generated in each Fenton system (Figure 1A-F) gave single peaks with a mean retention time of 17.3 ( 0.3 min. Figure 3B shows a chromatogram of spot 2 from the copper(II) Fenton system. Despite minor fluctuations in retention times due to subtle variations in performance of the

reverse phase column and solvent gradient, cochromatography experiments demonstrated that spot 1 generated in each Fenton system coeluted with one another to produce single peaks with a mean retention time of 19.0 ( 0.4 min. Figure 3C demonstrates cochromatography of spot 1 from the copper(II) Fenton system with spot 1 from the vanadium(III) Fenton system. Similarly, spot 2 generated in each Fenton system coeluted with one another, producing single peaks with a mean retention


Figure 3. HPLC chromatograms of 32P-labeled DNA lesions generated following exposure of salmon sperm DNA or dinucleotides to transition metal ion/hydrogen peroxide Fenton systems. Phenanthrene-9,10-diol, detected by its UV absorbance at 254 nm and having a retention time of approximately 40 min, was used as an internal standard in each case and is indicated by the dotted line trace. (A) Spot 1 from copper(II) Fenton system (Figure 1A); (B) spot 2 from copper(II) Fenton system (Figure 1A); (C) cochromatography of spot 1 from copper(II) Fenton system (Figure 1A) with spot 1 from vanadium(III) Fenton system (Figure 1F); (D) cochromatography of spot 2 from copper(II) Fenton system (Figure 1A) with spot 2 from cobalt(II) Fenton system (Figure 1C); (E) cochromatography of spot 1 from copper(II) Fenton system (Figure 1A) with dApdG spot 1 from copper(II) Fenton system (Figure 1K); (F) cochromatography of spot 2 from copper(II) Fenton system (Figure 1A) with dApdA spot 2 from copper(II) Fenton system (Figure 1L).

time of 16.9 ( 0.6 min, and an example demonstrating cochromatography of spot 2 from the copper(II) and cobalt(II) Fenton systems is shown in Figure 3D. Previous work in our laboratory suggested that the major bulky DNA lesions produced in DNA exposed to Cu2+/H2O2 were the result of an intrastrand cross-link between adjacent purines on the DNA sequence (29). In order to determine whether the same purine dimers could be formed in Fenton reactions mediated by the other transition metal ions, incubations using the purine dinucleotides dApdG and dApdA in place of DNA were performed. Subsequent 32P-postlabeling resulted in the detection of radiolabeled species with the same chromatographic mobility on anion-exchange TLC to spots 1 and 2 generated in DNA (Figure 1K,L). HPLC cochromatography experiments confirmed that spot 1 generated in DNA was the same as the spot obtained in all the Fenton incubations with dApdG. In each case, a single peak was obtained with a mean retention time of 18.7 ( 0.1 min [Figure 3E shows cochromatography of spot 1 and dApdG exposed to a copper(II) Fenton system]. Cochromatography also demonstrated that spot 2 generated in DNA was the same as the spot produced in each Fenton incubation with dApdA. In each case, single peaks were produced with a mean retention time of 17.6 ( 0.1 min, and an example demonstrating cochromatog-


Figure 4. Agarose gel electrophoresis of salmon sperm DNA treated with Fenton-type hydroxyl radical-generating systems. Lanes 1 and 13 show a 1 kb molecular weight marker; visible bands in the region indicated represent specific fragments of DNA as follows: 2036 bp, 1636 bp, 1018 bp, 506 bp, 396 bp, 344 bp, 298 bp, 220 bp, 201 bp, 154 bp, 134 bp, 75 bp. Lane 2 shows DNA treated with 50 mM hydrogen peroxide only while lane 12 shows untreated salmon sperm DNA. The remaining lanes show DNA treated with 50 mM hydrogen peroxide plus the following: lane 3, 250 µM copper(II) sulfate; lane 4, 1 mM sodium chromate(VI); lane 5, 800 µM cobalt(II) sulfate; lane 6, 150 µM iron(II) sulfate; lane 7, 1 mM nickel(II) sulfate; lane 8, 150 µM vanadium(III) chloride; lane 9, 1 mM cadmium(II) chloride; lane 10, 1 mM chromic(III) chloride; lane 11, 1 mM zinc(II) chloride.

raphy of spot 2 and dApdA treated with copper(II) and hydrogen peroxide is given in Figure 3F. Agarose Gel Electrophoresis. Agarose gel electrophoresis was carried out in order to determine whether the degradation of DNA occurred to an extent that would significantly reduce the formation of the intrastrand DNA lesions. Figure 4 shows an agarose gel following electrophoresis of DNA exposed to each of the Fenton systems at the metal ion concentration where formation of bulky DNA lesions was highest. In the Fenton systems which did not generate bulky lesions, samples of DNA exposed to Fenton systems involving 1 mM transition metal compound were analyzed. Lanes 1 and 13 in the gel contain a molecular weight marker, with visible bands of DNA fragments between 2036 and 75 base pairs, while lane 12 contains untreated salmon sperm DNA. Lane 2 shows DNA exposed to hydrogen peroxide only. The presence of a smear of DNA, with fragment size between approximately 200 and over 2000 base pairs, demonstrated that little strand breakage occurred in this control sample. Treatment of DNA with the copper(II) Fenton system (lane 3) and the chromium(III) Fenton system (lane 10) resulted in DNA degradation so extensive that no ethidium bromide-stained material was visible in the gel. Lanes 4 and 9, containing DNA exposed to the chromate(VI) and cadmium(II) Fenton system, exhibited a little strand breakage resulting in DNA fragments of between 75 and 1500 base pairs. The cobalt(II), nickel(II), and zinc(II) Fenton systems (lanes 5, 7, and 11, respectively) did not generate detectable strand breakage. In contrast, extensive strand breakage was evident with the iron(II) Fenton system (lane 6) and the vanadium(III) Fenton system (lane 8) where DNA fragment size was reduced to between 75 and 150 base pairs. DNA treated in the copper(II), iron(II), vanadium(III), and chromium(III) Fenton reactions could not be pre-


cipitated and hence was treated with the iminodiacetic acid affinity matrix prior to analysis by electrophoresis and postlabeling; the extensive DNA degradation demonstrated in these samples may explain the inefficient DNA precipitation.

Discussion The formation in DNA exposed to ROS of lesions with the chromatographic characteristics of DNA adducts formed by bulky carcinogens was first reported by Randerath et al. (28) and subsequently, but independently, by ourselves (29). In our study, we also demonstrated that the lesions were formed in single-stranded DNA, and, suspecting the formation of some form of intrastrand cross-link, we examined the products of a series of specific dinucleotides in Fenton-type reactions. These investigations revealed that of the two major lesions formed in DNA, one was also formed by the dipurine dApdA and the other was formed by the dipurine dApdG. We therefore proposed that these species with aromatic adduct-like characteristics are the result of the intrastand linking of specific adjacent bases in DNA (29). More recently, Randerath et al. (30) have made similar proposals concerning the general nature of these lesions on the basis of evidence from experiments with oligonucleotides containing defined dinucleotide sequences. In addition to the base-base interactions that we proposed, some of the lesions may be a consequence of base-sugar crosslinks. In other studies, some of these lesions have been detected in the tissues of animals treated with nickel or ferric salts (36, 37). The exact nature of these putative intrastrand cross-links remains to be determined. The hydroxyl radical is considered to be more damaging both in vitro and in vivo than other ROS including singlet oxygen, hydrogen peroxide, and superoxide (38). DNA is particularly susceptible to attack by the hydroxyl radical, and it is thought that mechanisms exist that allow the formation of this potent electrophile very close to the negatively charged DNA (13). One such mechanism is thought to be the Fenton reaction involving the association of a transition metal ion with part of the double helix, thus allowing the reduction of hydrogen peroxide and generation of the hydroxyl radical directly adjacent to the DNA (13). Small quantities of Fenton reagents are known to occur naturally in vivo. For example, hydrogen peroxide is a common metabolite in aerobic cells whose intracellular concentration varies between different tissues (38). In addition, it can be overproduced in a number of disease states and at sites of inflammation (4). Trace elements, particularly transition metals, are also present in cells. Copper and iron, for example, are both present in blood plasma as metalloproteins and as a number of transport and storage complexes, and they can become available for Fenton chemistry in vivo (38). Other transition metals are less abundant in mammalian cells; however, occupational exposure has been shown to result in greatly enhanced levels in human biopsy tissues (39). Nickel, chromium, cobalt, and vanadium compounds can be ingested in various forms via pulmonary, oral, or dermal routes(40, 41, 42). Subsequent “facultative phagocytosis” by cells of particulate metal compounds, such as nickel, and then solubilization by lysozymes can result in the delivery of a very large amount of transition metal ions into the cytoplasm (40). Oxidative damage in cells following this phagocytic process is thought to result

Lloyd et al. Table 2. Comparison of the Total Formation of Bulky Oxidative Lesions and Generation of Strand Breaks in DNA Mediated by Fenton Reactions transition metal ion

maximum total generation of bulky oxidative lesions per 108 nucleotides

strand breaks

copper(II) chromium(VI) cobalt(II) iron(II) nickel(II) vanadium(III) cadmium(II) chromium(III) zinc(II)

75.6 25.1 47.5 21.7 26.2 17.1 -a -

+++ + ++ ++ + +++ -

a Failure to detect bulky lesions may have been due to inhibition of the 32P-postlabeling reaction by residual Cr(III) ions. See text for details.

either from redox cycling or from a respiratory burst induced by the phagocytic action of the cell (40). Oxidative damage can also arise during the reduction of transition metal ions in the cell. Chromium(VI), for example, is the only oxidation state of chromium that commonly crosses cell membranes (43). Once inside the cell, it is rapidly reduced by cellular chemicals such as ascorbate and glutathione, producing reactive intermediates such as chromium(V), singlet oxygen, and the hydroxyl radical. Reduction of transition metal ions to lower oxidation states, for example, chromium(V), can result in further catalysis of the Fenton reaction and is likely to increase levels of the hydroxyl radical in cells (43). The difficulty in estimating physiological concentrations of Fenton reagents has meant that the extent to which the Fenton reaction occurs in vivo is still a matter of some debate. It has been argued that the low concentrations of transition metal ions and hydrogen peroxide in cells render the reaction too slow to be biologically significant (44, 45). However, the epidemiological evidence regarding occupational exposure to transition metals and the transport mechanisms available to them suggests that significant amounts might become available for Fenton chemistry. Concentrations of Fenton reagents may vary between different tissues in the body, and their local concentrations, particularly in the region of chromosomes, could be much higher than elsewhere in the cell. In addition, a number of human diseases characteristically result in intracellular accumulation of high levels of metal ions, including copper (Wilson’s disease) and iron (primary hemochromatosis). Indeed, DNA lesions similar to those generated in this study have been detected in the livers of patients with these diseases (46). In the present study, we have demonstrated the generation of both strand breaks and bulky lesions following exposure of DNA to Fenton-type radicalgenerating reactions involving a number of transition metal compounds. The strand breaks could be either true double-strand breaks or single-strand breaks generated at the same position in each strand of DNA. The extent to which the nine transition metal ions tested mediate oxidative DNA damage in Fenton reactions is summarized in Table 2. The results demonstrate a lack of correlation between generation of bulky DNA lesions and strand breaks. Although the copper(II)/hydrogen peroxide system produced the highest yield of bulky lesions, this system also generated the highest degree of strand breakage, resulting in the production of DNA fragments


of less than 75 base pairs. Similarly, the iron and vanadium Fenton systems resulted in extensive DNA strand breakage at higher metal ion concentrations. The high level of DNA degradation in these three Fenton reactions probably explains the reduction in yield of the bulky lesions at higher concentrations of these transition metal ions. Indeed, DNAs treated in the iron and vanadium Fenton systems, digested to nucleosides and analyzed by liquid chromatography, exhibit such extensive DNA damage that no intact nucleosides are present at the higher metal ion concentrations (data not shown). In contrast, the cobalt, nickel, and chromium(VI) Fenton systems resulted in little or no detectable strand breakage, while the yield of bulky adducts remained moderately high. Exposure of DNA to cadmium, chromium(III), and zinc Fenton systems did not result in the generation of significant levels of bulky DNA lesions; however, DNA strand breaks were evident in the cadmium and particularly the chromium(III) Fenton systems. This is in agreement with results from other studies demonstrating oxidative damage in DNA treated with these transition metal ions (47, 48), and suggests that they are able to take part in Fenton-type reactions that generate the hydroxyl radical. In contrast, zinc(II) is regarded as Fenton-inactive due to its fixed valency and was the only transition metal ion tested here that exhibited no detectable oxidative DNA damage. An interesting observation from the postlabeling analysis is that each transition metal ion produced different proportions of each bulky lesion (Table 1). Indeed, the copper(II) and vanadium(III) Fenton reactions generated significant levels of bulky lesions in DNA that were unique to those systems. The ability of different transition metal ions to generate differing levels of bulky DNA lesions might suggest that the production of these lesions involves an interaction between the metal ion and DNA. It also suggests that these interactions may differ between metal ions, resulting in the generation of differing levels of DNA lesions. A limitation of the postlabeling assay is its acute sensitivity to small amounts of contaminants in the DNA sample. Hence, when interpreting the data in the present study, there was concern regarding the efficiency of DNA purification, particularly with regard to removal of trace metal ions. Of the nine transition metal ions used in these experiments, all but one demonstrated little inhibition of the postlabeling assay (typically around 60%) following addition of 1 mM transition metal ions and subsequent precipitation of the DNA or treatment with the chelating agent. However, residual chromium(III) in PAH-modified DNA almost completely inhibited the labeling of PAH-DNA adducts; hence, we cannot state unequivocally that no bulky lesions were generated in DNA treated with the chromium(III) Fenton system. Indeed, the actual levels of bulky oxidative lesions generated in the other Fenton systems may be significantly higher than reported here. However, the apparent similarity in the ability of residual transition metal ions to inhibit the postlabeling assay demonstrates that the levels of bulky DNA lesions quoted in the present study are at least representative of the absolute levels. Furthermore, it must be borne in mind that 1 mM of each transition metal ion was used in these control experiment and this was the highest concentration used in the Fenton experiments; hence, at the lower concentrations of transition metal ions used in these experiments, the degree of inhibition is likely to be much less.


To date, no hypothesis as to a possible mechanism of formation of dimeric bulky DNA lesions, such as those described here, has been put forward. The ability of different transition metal ions to associate with either DNA bases or phosphate groups (49) may explain why the transition metal ions tested in the present study generate different levels of bulky oxidative lesions and strand breaks. In view of the evidence presented here that generation of the putative intrastrand cross-links in DNA does not correlate with strand breaks, we propose that the production of these two types of oxidative damage arises via two different mechanisms, perhaps involving the association of transition metal ions with different regions of the double helix and different moieties of individual nucleotides. The formation of the two types of oxidative DNA damage described in the present study and their relationship with the generation of other types of oxidative damage will be explored further.

Acknowledgment. D.R.L. gratefully acknowledges receipt of a Research Studentship from the Institute of Cancer Research.

References (1) Gutteridge, J. M. (1994) Biological origin of free radicals, and mechanisms of antioxidant protection. Chem.-Biol. Interact. 91, 133-140. (2) Constantin, D., Mehrotra, K., Moldeus, P., and Jernstrom, B. (1995) Studies on the effect of sulfite on benzo(a)pyrene-7,8dihydrodiol activation to reactive intermediated in human polymorphonuclear leukocytes. Chem.-Biol. Interact. 94, 73-82. (3) Halpern, H. J., Yu, C., Barth, E., Peric, M., and Rosen, G. M. (1995) In situ detection, by spin trapping, of OH• markers produced from ionization radiation in the tumour of a living mouse. Proc. Natl. Acad. Sci. U.S.A. 92, 796-800. (4) Lewis, J. G., and Adams, D. O. (1987) Inflammation, oxidative DNA damage and carcinogenesis. Environ. Health Perspect. 76, 19-27. (5) Miesel, R., Zuber, M., Sanocka, D., Graetz, R., and Kroeger, H. (1994) Effects of allopurinol on in vivo suppression of arthritis in mice and ex vivo modulation of phagocytic production of oxygen radicals in whole human blood. Inflammation 18, 597-612. (6) Varma, S. D., Devamanoharan, P. S., and Morris, S. M. (1995) Prevention of cataracts by nutritional and metabolic antioxidants. Crit. Rev. Food Sci. Nutr. 35, 111-129. (7) Banki, K., Colombo, E., Sia, F., Halladay, D., Mattson, D. H., Tatum, A. H., Massa, P. T., Phillips, P. E., and Perl, A. (1994) Oligodendrocyte-specific expression and autoantigenicity of transaldolase in multiple sclerosis. J. Exp. Med. 180, 1649-1663. (8) Gutteridge, J. M. (1992) Ageing and free radicals. Med. Lab. Sci. 49, 313-318. (9) Feig, D. I., Reid, T. M., and Loeb, L. A. (1994) Reactive oxygen species in tumorigenesis. Cancer Res. 54, 1890s-1894s. (10) Carmichael, P. L., and Hipkiss, A. R. (1991) Differences in susceptibility between crystallins and non-lenticular proteins to copper and hydrogen peroxide-mediated peptide bond cleavage. Free Radical Res. Commun. 15, 101-110. (11) Halliwell, B., and Gutteridge, J. M. C. (1985) The importance of free radicals and catalytic metal ions in human disease. Mol. Aspects Med. 8, 89-193. (12) Cheeseman, K. H., Beavis, A., and Esterbauer, H. (1988) Hydroxyl radical-induced iron-catalysed degradation of 2-deoxyribose. Biochem. J. 252, 649-653. (13) Pryor, W. A. (1988) Why is the hydroxyl radical the only radical that commonly adds to DNA? Free Radical Biol. Med. 4, 219223. (14) Kasai, H., Crain, P. F., Kuchino, Y., Nishimura, S., Ootsuyama, A., and Tanooka, H. (1986) Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 7, 1849-1851. (15) Hegi, M. E., Sagelsdorff, P., and Lutz, W. K. (1989) Detection by 32P-postlabelling of thymidine glycol in γ-irradiated DNA. Carcinogenesis 10, 43-47. (16) Kawanishi, S., Inoue, S., and Sano, S. (1986) Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide. J. Biol. Chem. 261, 5952-5958.

400 Chem. Res. Toxicol., Vol. 10, No. 4, 1997 (17) Kawanishi, S., Inoue, S., and Yamamoto, K. (1989) Site-specific DNA damage induced by nickel(II) ion in the presence of hydrogen peroxide. Carcinogenesis 10, 2231-2235. (18) Shi, X., Mao, Y., Knapton, A. D., Ding, M., Rojanasakul, Y., Gannett, P. M., Dalal, N., and Liu, K. (1994) Reaction of Cr(VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(IV)-mediated Fenton-like reaction. Carcinogenesis 15, 2475-2478. (19) Reid, T. M., and Loeb, L. A. (1993) Tandem double CC f TT mutations are produced by reactive oxygen species. Proc. Natl. Acad. Sci. U.S.A. 90, 3904-3907. (20) Tkeshelashvili, L. K., Reid, T. M., McBride, T. J., and Loeb, L. A. (1993) Nickel induces a signature mutation for oxygen free radical damage. Cancer Res. 53, 4172-4174. (21) Du, M.-Q., Carmichael, P. L., and Phillips, D. H. (1994) Induction of activating mutations in the human c-Ha-ras-1 proto-oncogene by oxygen free radicals. Mol. Carcinog. 11, 170-175. (22) Fenton, H. J. H. (1894) Oxidation of tartaric acid in presence of iron. J. Chem. Soc., 899-910. (23) Kortenkamp, A., Ozolins, Z., Beyersmann, D., and O’Brien, P. (1989) Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione. Mutat. Res. 216, 19-26. (24) Fresco, P., and Kortenkamp, A. (1994) The formation of DNA cleaving species during the reduction of chromate by ascorbate. Carcinogenesis 15, 1773-1778. (25) Tachon, P. (1989) Ferric and cupric ions requirement for DNA single-strand breakage by hydrogen peroxide. Free Radical Res. Commun. 7, 1-10. (26) Lewis, C. P. L., Demedts, M., and Nemery, B. (1991) Indices of oxidative stress in hamster lung following exposure to cobalt(II) ions: in vivo and in vitro studies. Am. J. Respir. Cell. Mol. Biol. 5, 163-169. (27) Sakurai, H. (1994) Vanadium distribution in rats and DNA cleavage by vanadyl complex: implication for vanadium toxicity and biological effects. Environ. Health Perspect. 102, 35-36. (28) Randerath, K., Yang, P.-F., Danna, T. F., Reddy, R., Watson, W. P., and Randerath, E. (1991) Bulky adducts detected by 32Ppostlabelling in DNA modified by oxidative damage in vitro. Comparison with rat lung I-compounds. Mutat. Res. 250, 135144. (29) Carmichael, P. L., Ni She´, M., and Phillips, D. H. (1992) Detection and characterization by 32P-postlabelling of DNA adducts induced by a Fenton-type oxygen radical-generating system. Carcinogenesis 13, 1127-1135. (30) Randerath, K., Randerath, E., Smith, C. V., and Chang, J. (1996) Structural origins of bulky oxidative DNA adducts (type II I-compounds) as deduced by oxidation of oligonucleotides of known sequence. Chem. Res. Toxicol. 9, 247-254. (31) Giles, A. S., Seidel, A., and Phillips, D. H. (1995) In vitro reaction with DNA of the fjord-region diol epoxides of benzo[g]chrysene and benzo[c]phenanthrene as studied by 32P-postlabelling. Chem. Res. Toxicol. 8, 591-599. (32) Gupta, R. C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. U.S.A. 81, 6943-6947. (33) Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551.

Lloyd et al. (34) Pfau, W., and Phillips, D. H. (1991) Improved reverse phase highperformance liquid chromatographic separation of 32P-labelled nucleoside 3′,5′-bisphosphate adducts of polycyclic aromatic hydrocarbons. J. Chromatogr. 570, 65-76. (35) Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (36) Chang, J., Watson, W. P., Randerath, E., and Randerath, K. (1993) Bulky DNA adduct formation induced by Ni(II) in vitro and in vivo as assayed by 32P-postlabelling. Mutat. Res. 291, 147-159. (37) Randerath, E., Watson, W. P., Zhou, G.-D., Chang, J., and Randerath, K. (1995) Intesification and depletion of specific bulky renal DNA adducts (I-compounds) following exposure of male F344 rats to the renal carcinogen ferric nitriloacetate (Fe-NTA). Mutat. Res. 341, 265-279. (38) Halliwell, B., and Gutteridge, J. M. C. (1989) Free radicals in biology and medicine, 2nd ed., pp 29-31, pp 33-46, and pp 7991, Oxford University Press, Oxford, U.K. (39) IARC (1990) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Vol. 49, Chromium, nickel and welding, IARC, Lyon. (40) Costa, M. (1991) Molecular mechanisms of nickel carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 31, 321-337. (41) Le´onard, A., and Lauwerys, R. (1990) Mutagenicity, carcinogenicity, and teratogenicity of cobalt metal and cobalt compounds. Mutat. Res. 239, 17-27. (42) Nechay, B. R., Nanninga, L. B., and Nechay, P. S. E. (1986) Vanadyl(IV) and vanadate(V) binding to selected endogenous phosphate, carboxyl, and amino ligands; calculations of cellular vanadium species distribution. Arch. Biochem. Biophys. 251, 128138. (43) Le´onard, A., and Lauwerys, R. R. (1980) Carcinogenicity and mutagenicity of chromium. Mutat. Res. 76, 227-239. (44) Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Is hydroxyl radical generated by the Fenton reaction in vivo? Biochem. Biophys. Res. Commun. 130, 533-539. (45) Fee, J. A. (1982) Is superoxide important in oxygen poisoning? Trends Biochem. Sci. 7, 84-86. (46) Carmichael, P. L., Hewer, A., Osborne, M. R., Strain, A. J., and Phillips, D. H. (1995) Detection of bulky DNA lesions in the liver of patients with Wilson’s disease and primary haemochromatosis. Mutat. Res. 326, 235-243. (47) Littlefield, N. A., and Hass, B. S. (1995) Damage to DNA by cadmium or nickel in the presence of ascorbate. Ann. Clin. Lab. Sci. 25, 485-492. (48) Tsou, T. C., Chen, C. L., Liu, T. Y., and Yang, J. L. (1996) Induction of 8-hydroxydeoxyguanosine in DNA by chromium(III) plus hydrogen peroxide and its prevention by scavengers. Carcinogenesis 17, 103-108. (49) Eichhorn, G. L., and Shin, Y. A. (1968) Interaction of metal ions with polynucleotides and related compounds. XII. The relative effect of various metal ions on DNA helicity. J. Am. Chem. Soc. 90, 7323-7328.

TX960158Q

Generation of Putative Intrastrand Cross-Links and Strand Breaks in

Recommend Documents