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Induction of oxidative DNA damage by carcinogenic metals
Toxicology Letters 127 (2002) 55 – 62 www.elsevier.com/locate/toxlet
Induction of oxidative DNA damage by carcinogenic metals Wojciech Bal a,b,*, Kazimierz S. Kasprzak c a
Faculty of Chemistry, Uni6ersity of Wroclaw, Ul. F. Joliot-Curie 14, 50 -383 Wroclaw, Poland b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland c Laboratory of Comparati6e Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD, USA
Abstract The metal ions carcinogenic to humans are As, Be, Cd, Cr and Ni, and the candidates also include Co, Cu, Fe and Pt. A range of molecular mechanisms was proposed for these metals, reflecting their diverse chemical properties. The oxidative concept in metal carcinogenesis proposes that some complexes of the above metals (Co, Cr, Cu, Fe, Ni) formed in vivo undergo redox cycling, yielding reactive oxygen species and/or high valence metal ions which oxidize DNA. Some of the products of oxidative DNA damage, including 8-oxoguanine and strand breaks, induce mutations, which may lead to neoplastic transformation. The establishment of metal-binding modes in the cell nucleus and of their reactivity is crucial for the understanding of molecular events in metal carcinogenesis. We have proposed the binding sites for Ni(II) and Cu(II) in core histones (H3, H2A) and sperm protamines (HP2) and, using molecular models, provided evidence for the generation of promutagenic oxidative DNA damage by the bound metals. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carcinogenesis; DNA; Histones; Metals; Oxidative damage; Oxygen radicals
1. Introduction The list of metals and metalloids whose compounds are definitely carcinogenic to humans includes (in alphabetical order) As, Be, Cd, Cr and Ni. There are more candidate elements emerging from current research, including Co, Cu, Fe and Pt (Kasprzak and Buzard, 2000). These elements are spread all over the periodic table, and, accordingly, their chemical properties are quite diverse. Therefore, a range of molecular mechanisms has been proposed for these metals. * Corresponding author. Tel.: + 48-71-3757281; fax: +4871-3282348. E-mail address: [email protected] (W. Bal).
The oxidative concept in metal carcinogenesis proposes that complexes formed by some of the above metals (Co, Cr, Cu, Fe, Ni) in vivo in the vicinity of DNA catalyze redox reactions, whose products oxidize DNA. In turn, some of the products of DNA-oxidative damage induce mutations, which may lead to neoplastic transformation. Such products include modified bases (primarily 8-oxoguanine), abasic sites (primarily depurinated), base adducts of carbon-centered radicals (including DNA-crosslinked proteins), and single and double breaks in the phospho-sugar backbone of DNA (Aust and Eveleigh, 1999). Two important distinctions have to be made within the list of oxidative carcinogenic metals. The first one is to separate chromium from the
0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 4 8 3 - 0
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others. Its uniqueness stems from its strict assignment of carcinogenicity to one level of oxidation, namely +6 (which is not accessible to other carcinogenic metals), as opposed to the physiologically essential, although still enigmatic Cr(III) as part of the glucose tolerance factor (Vincent, 1999). The other type of distinction, between Ni and the tentative carcinogens Co, Cu and Fe, is physiological rather than chemical. These metals are essential microelements with multiple physiological functions, and thus with specific endogenous transport and control mechanisms. Ni has also been named essential for mammals (Spears, 1984). However, no specific physiological functions have ever been found for it, and the specific character of respective experiments leads to a speculation that the positive result reflects the requirements of the bacteria of the digestive tract of experimental animals rather than the animals themselves.
2. Molecular mechanisms of chromium carcinogenesis Because of its uniqueness, chromium will be discussed first. As mentioned above, this metal is carcinogenic when administered as anionic chromate or dichromate [Cr(VI)], and not as a Cr(III) species, because Cr(VI) anions are much more easily taken up by cells via physiological sulfate and phosphate transporters (Stearns and Wetterhahn, 1997). These anions cannot interact with DNA in vitro, because of mutual electrostatic repulsion. The intracellular Cr(VI) is reduced by a variety of factors, including glutathione (GSH), glutathione reductase, carbohydrates and ascorbic acid. It is paradoxical that the carcinogenicity of chromates is induced by cellular defense systems poised against the oxidative assault. Many not yet fully characterized short-lived reactive species formed in this way contain Cr(V) and Cr(IV). The chromate reductions can either be one-electron reactions, e.g. Cr(VI)Cr(V), Cr(V) Cr(IV), or two-electron reactions, e.g. Cr(VI) Cr(IV), Cr(V) Cr(III), depending on the reductant. Oxygen, carbon and sulfur-centered radicals, formed during the reduction, attack and damage DNA
and other cellular components. The DNA damage includes base oxidation, depurination (guanine) and DNA cross-links with proteins and radical products of cellular reductants (Kasprzak, 1996; Kasprzak and Buzard, 2000; Sugden and Stearns, 2000). The final product of chromate reduction, Cr(III), is also involved in the cross-link formation and, perhaps, in causing base substitution mutations, through non-redox mechanisms (Tsou et al., 1997). It seems that Cr(III) can also maintain redox activity in vivo through Cr(III)/Cr(V) (Dillon et al., 2000) or Cr(III)/Cr(II) redox pairs (Speetjens et al., 1999), thus re-activating the radical cascade. 3. Mechanisms of the formation of radical species The remaining metals (Co, Cu, Fe, Ni) in their physiologically relevant forms are predominantly, although not exclusively, one-electron reactants (Kasprzak, 1996; Landolph, 1999). The general mechanism of oxygen activation by such metal ions is provided by Fenton/Haber –Weiss and autoxidation reactions, in which molecular oxygen and its and H2O2 partially reduced forms superoxide O− 2 serve as substrates. Reactions (1) and (2) present the conversion of H2O2 into hydroxyl radical (OH) via the oxidation of a metal cation (note that the metal species serves as a reducing agent): Mn + + H2O2 M(n + 1) + + OH − + OH (Fenton reaction)
(1)
The redox cycling of the metal cation is maintained by reduction with O− 2 : (2) M(n + 1) + + O2− Mn + + O2 The balance of these two reactions is: H2O2 + O2− O2 + OH − + OH (Haber–Weiss reaction) (3) The only requirement for the above scheme of metal ion-catalyzed formation of hydroxyl radicals from unavoidable by-products of oxygen metabolism (Sohal and Dubey, 1994) is that a given metal ion has two neighboring oxidation states (Mn + and M(n + 1) + ) available at the same
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conditions. In the absence of chelators, the Fenton reaction is driven by Cu(I), Fe(II) and Co(II), but not by Ni(II) aqua ions. It is, however, unrealistic to expect unbound metal species under cellular conditions, in the presence of a plethora of proteins, and millimolar concentrations of such cellular components as glutathione, ascorbate, other carboxylic acids and nucleotides. Chelation, affecting the redox potentials of metal ions, modulates their ability to participate in the redox cycle. The oxidized form of the metal can also originate from auto-oxidation in the presence of molecular oxygen: Mn + +O2 M(n + 1) + +O2− M
n+
(4)
+O +2H+ M(n + 1) + +H2O2 − 2
(5)
The occurrence of the above reactions as well depends strongly on the presence or absence of specific chelators. Eqs. (2) and (5) demonstrate that, depending on the reaction conditions, O− 2 may be either the reductant or oxidant for a metal ion. Metal complexes can also be involved in twoelectron reactions, which yield strong oxidants alternative to OH, such as oxo and peroxo species, and singlet oxygen (in a decomposition process). Mn + +H2O2 [MO]n + +H2O n+
M
+H2O2 [MO2]
(n − 2) +
+2H
(6) +
(7)
It is often difficult to differentiate between the free and metal-bound oxygen radical species. Each case is specific and requires elaborate studies. For example, it has been established that metal-bound oxygen species are formed only in the reaction of Cu(I) aqua ion with H2O2 (Masarwa et al., 1988), while the EDTA complex of Fe(II) catalyzes H2O2 decomposition to free OH radicals (Tullius, 1987; Celender and Cech, 1990).
4. Oxidative chemistry of nickel(II) The ‘uncomplexed’ Ni(II) does not activate H2O2 (Cotelle et al., 1992; Shi et al., 1993) and is
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only weakly bound, and generally innocent to double-stranded DNA (Lee et al., 1982; Kasprzak et al., 1986). On the other hand, the effects of Ni(II) on DNA in vivo suggest Fenton-like behavior (Tkeshelashvili et al., 1993). In chemical studies on potential ligands that might facilitate nickel’s oxidative activity it has been established that only low-spin Ni(II) complexes (mainly of square-planar structure) can be oxidized to Ni(III) in water solution (Lancaster, 1988). Quite interestingly, this is a common feature of Ni(II) complexes with oligopeptides, even as simple as tetraglycine (Bossu et al., 1978). However, for most of the peptides, the appropriate complexes are formed at high, non-physiological pH values above 9 (Sigel and Martin, 1982). The relevant exclusion from this rule is provided by peptides having a His residue at position 3, which form low-spin, square-planar complexes at physiological pH (Kozłowski et al., 1999). The simplest of such peptides, Gly-Gly-His, has been studied, and was indeed found to activate not only hydrogen peroxide, but also the molecular oxygen in ambient air (Bossu and Margerum, 1976). The subsequent work has, however, demonstrated that this high reactivity is a consequence of the presence of a free terminal His carboxylate (Bal et al., 1994), which can also convey carbon radical reactions (Burrows et al., 1998). The peptides with the modified carboxylate function (tripeptide amides or longer peptides) require H2O2 or other oxidants (e.g. oxone). Nevertheless, Ni(II) complexes of Gly-Gly-His, its analogues with positions 1 and 2 modified (Xaa-Yaa-His in general) (Liang et al., 1995, 1998) and DNA binding proteins, extended N-terminally with the Gly-Gly-His sequence have gained interest as potential specific DNA cleavage agents in the presence of oxidants (Mack and Dervan, 1990; Harford et al., 1996). Another approach is to design non-peptidic macrocyclic Ni(II) complexes as DNA damaging agents (Muller et al., 1999). This approach has allowed studies of covalent adduct formation (alkylation) at nucleobases, because the macrocycles can be designed so that their oxidation potentials have values similar to those of the central ion, and thus the intermediate radical species can be localized on the ligand rather than on the metal ion.
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5. Formation of oxidizing species in Ni(II) interactions with histones The above studies, although providing important insights into the potential mechanisms of DNA damage by nickel species, and perhaps having future practical applications, still do not answer the question of actual ligands for Ni(II) in vivo, which might turn it into a Fenton-like agent. In search of such ligands we turned our interest to histones, as the most abundant proteins of cell nucleus (with formal concentrations of ca. 3 mM) (Bal et al., 1999). The initial studies demonstrated that nuclear proteins promote DNA oxidation by Ni(II) and H2O2 (Kasprzak and Bare, 1989; Nackerdien et al., 1991) and that nucleohistone markedly enhances 8-oxo-dG formation by the same system (Datta et al., 1993). Our analysis of sequences of histones, reviewed previously (Bal and Kasprzak, 1997; Bal et al., 2000a) and confirmed by the published three-dimensional structure of nucleosome (Luger et al., 1997) indicated only two potential binding sites for Ni(II), one in histone H3 ( –CAIH – motif in positions 110– 113), and another in histone H2A (– ESHH – motif in positions 121– 124). We studied them using oligopeptide models and partial protein assemblies. The study of the – CAIH – motif revealed the formation of a low-spin complex with the oligopeptide as well as with the histone core tetramer in vitro, and demonstrated that such a complex facilitates the 2%-deoxyguanosine oxidation in the presence of H2O2 (Bal et al., 1995, 1996, 1999). The binding constant obtained for the histone tetramer was relatively high (log K of 4.26 –5.26, depending on the extent of protein aggregation). However, auto-oxidation of the peptide complex to a disulfide was detected, which diminished the binding constant substantially. In the studies of Ni(II) binding to the oligopeptide derived from histone H2A, – TESHHK – , we established that the initial complex formed at physiological pH is high-spin, and, quite expectably, does not activate hydrogen peroxide (Bal et al., 1998, 2000b). The binding affinity of H2A for Ni(II) is comparable to that of H3 (Bal et al., 2000a). However, binding of Ni(II) to the –TESHHK – domain, studied using this hexapep-
tide, as well as the 34-peptide representing the whole C-terminal domain of H2A, and the whole histone H2A, resulted in the hydrolysis of the Glu-Ser peptide bond, with the formation of Ni(II) complexes with the – SHHK – motif of the cut-off peptides (i.e. SHHK-amide or SHHKAKGK, respectively). The Ni(II) complex thus formed demonstrated, not surprisingly, oxidative abilities in the presence of H2O2, cleaving the plasmid DNA, and oxidizing nucleobases. Oxidation of the peptide was also observed. Summarizing this line of research, we can conclude that our model peptide studies provided the— so far unique— mechanism of formation of a square-planar Ni(II) complex, derived from the core histone. Such a complex, capable of participating in Fenton chemistry, is a good candidate for an in vivo agent of DNA oxidative damage. At this juncture, our studies also indicated that cysteine peptides, such as –CAIH – from histone H3, might provide an alternative to His-3 ligands.
6. Metal –protamine interactions Protamines are highly basic peptides that substitute for histones in the sperm cell nuclei. Sperm DNA appears to be particularly susceptible to oxidative assault, because the oxidative metabolism is particularly active in the sperm, and yet there are no DNA repair mechanisms in the highly compacted sperm chromatin (Pogany et al., 1981). This disadvantage is compensated by the abundance of copper–zinc superoxide dismutase, an enzyme protecting from the superoxide radicals that leak from the mitochondria (Gu et al., 1995). The tentative relationship between the sperm and nickel carcinogenesis is provided by the concept of paternally transmitted transgenerational carcinogenesis, applied to the epidemiology of cancer incidence in the progeny of fathers occupationally exposed to heavy metals (Anderson et al., 1994; Buckley, 1994). Mammalian protamines belong to two families: P1 and P2. P1 is expressed in all species, while P2 in substantial amounts is present in just a few species, including human, horse and mouse (McKay et al., 1986). In these species, P2 is
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absolutely necessary for fertility (Belokopytova et al., 1993). It was therefore very interesting to find out that human P2 (HP2) is a His-3 peptide. We undertook the study of Ni(II) and Cu(II) binding to HP2 and the oxidative properties of resulting complexes. To facilitate the studies, we used the N-terminal 15-peptide of HP2 (HP21 – 15) as a model. The results (Bal et al., 1997a,b; Liang et al., 1999) illustrated the dual role of HP21 – 15 as a chelator: it quenched the DNA strand-breaking ability of Cu(II) in the presence of H2O2, but activated that of Ni(II). In contrast to Ni(II), the Cu(II)–H2O2 system without HP21-15 generates strand breaks and base oxidation in doublestranded DNA efficiently (Yamamoto and Kawanishi, 1989) (see below). The structural basis for the quenching was provided in a subsequent NMR study. It revealed a specific structure assumed by the HP21 – 15 peptide upon metal binding, with the Tyr-8 ring, placed in the vicinity of the metal site, serving as a potential suicide target for radical attack (Bal et al., 2000c). The likely explanation for the differences of properties between Ni(II) and Cu(II) complexes is a different nature of the reactive oxygen species produced by each metal: a metal-bound one for Cu(II), similar to that found for the Cu(II) aqua ion– DNA interaction (Yamamoto and Kawanishi, 1989), and a diffusible one for Ni(II). The role of HP2 as a protectant against adventitious copper, and at the same time as an activator of another metal, possibly responsible for nickel-related sperm damage, emerged from these studies.
7. DNA oxidation by metal complexes The protamine results lead to copper and other essential metals as potential mediators of oxidative DNA damage. Until recently, the essential metals, copper and iron, were not considered as agents of oxidative DNA damage in vivo, because they are under strict physiological control during uptake, transport and metabolism. However, there is an emerging view that this control may not be sufficient in certain pathological situations, such as inflammation, intoxication, or intense oxidative metabolism, which may damage mitochon-
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dria and overwhelm the antioxidant defense systems. The essential metals might then become local (ultimate) carcinogens, as suggested for copper and hepatic hyperplasia (Eagon et al., 1999). The spectra of the mutational damage of free or chromatin DNA exposed to iron, copper and cobalt are similar to each other (and to nickel in chromatin). This finding supports the notion of a common underlying mechanism, namely hydroxyl radical-like attack (Tkeshelashvili et al., 1991, 1993; Rodriguez et al., 1997; Lloyd et al., 1997). Nonetheless, quantitative differences between individual agents have been reported, reflecting the diversity of specific mechanisms, e.g. proportion of base damage to strand breaks (apparently low for diffusible radicals and high for metal-bound oxidants) (Kennedy et al., 1997). In fact, the search for potential ligands in vivo for these metals is still just beginning. Basic facts on the reactivities of various model chelators in vitro and in vivo are being established. Like in the case of nickel, the mode of chelation is decisive for the activity, although most of the systems reported are active in vitro. For iron, the formation of Fe(II)/Fe(III) redox pair is beyond doubt. Positive examples supporting its DNA damaging activity include EDTA and NTA as ligands. These two are not very discriminate between Fe(II) and Fe(III). However, iron complexes with o-phenanthroline and deferoxamine, which strongly stabilize Fe(II), and with citric acid, stabilizing Fe(III), are inactive (Landolph, 1999), the latter in vivo rather than in vitro (Hartwig and Schlepegrell, 1995). For copper, the formation of Cu(I)/Cu(II) redox pair is usually invoked. This is certainly true for the interaction of copper with DNA in the absence of other bioligands (Yamamoto and Kawanishi, 1989; Drouin et al., 1996) and for some well-established chelators, such as ophenanthroline (Meijler et al., 1997). On the other hand, our recent research on the copper complexes of aminoglycoside antibiotics indicated that both Cu(I)/Cu(II) and Cu(II)/Cu(III) redox couples may be accessible for the same bioligand (Jez; owska-Bojczuk et al., 2001a,b). Finally, in a study of Cu(II) binding to the – TESHHK – model of histone H2A we found not only specific complex formation, but also strong oxidative ac-
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tivity in the presence of H2O2, which involved the Cu(II)/Cu(III) redox system (Mylonas et al., 2001). In contrast with the nickel study, the hydrolysis was not required for this oxidative activity. The latter work indicates that adventitious copper may also find docking sites in nuclear proteins, where it can exert site-specific damage.
8. Conclusion This brief and necessarily incomplete review of essential metals as potential carcinogenic DNA oxidants indicates that individual mechanisms are controlled by the local availability of specific ligands. We would also like to highlight the competition for metal ions between target proteins and/or DNA on one site and cellular low molecular weight ligands on the other. The establishment of tentative affinity constants for nickel complexes with histones allowed for calculations indicating that at high intracellular GSH levels, Ni(II) binding to histones may be negligibly low (Bal et al., 1999, 2000a). Formation of such complexes is possible at GSH concentrations lower than 1 mM, which correlates with the effects of Ni(II) seen in GSH-challenged cell cultures (Lynn et al., 1994; Salnikow et al., 1994). Such results will undoubtedly have to be taken into account in search for the mechanisms of oxidative DNA damage by carcinogenic metals.
Acknowledgements Dr W. Bal acknowledges the financial support from the Alexander von Humboldt Foundation.
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Jez; owska-Bojczuk, M., Les´niak, W., Bal, W., Kozłowski, H, Gatner, K., Jezierski, A., Sobczak, J., Mangani, S., MeyerKlaucke, W., 2001b. Molecular mechanism of hydrogen peroxide conversion and activation by Cu(II)-amikacin complexes. Chem. Res. Toxicol. 14, 1353 – 1362. Kasprzak, K.S., 1996. Oxidative DNA damage in metal-induced carcinogenesis. In: Chang, L.W., Magos, L., Suzuki, T. (Eds.), Toxicology of Metals. Lewis Publishers, Boca Raton, pp. 299 – 320. Kasprzak, K.S., Bare, R.M., 1989. In 6itro polymerization of histones by carcinogenic nickel compounds. Carcinogenesis 10, 621 – 624. Kasprzak, K.S., Buzard, G.S., 2000. The role of metals in oxidative damage and redox cell signaling derangement. In: Koropatnick, J., Zalups, R. (Eds.), Molecular Biology and Toxicology of Metals. Taylor and Francis, London, pp. 477 – 527. Kasprzak, K.S., Waalkes, M.P., Poirier, L.A., 1986. Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in 6itro. Toxicol. Appl. Pharmacol. 82, 336 – 343. Kennedy, L.J., Moore, K. Jr., Caulfield, J.L., Tannenbaum, S.R., Dedon, P.C., 1997. Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents. Chem. Res. Toxicol. 10, 386 – 392. Kozłowski, H., Bal, W., Dyba, M., Kowalik-Jankowska, T., 1999. Specific structure – stability relations in metallopeptides. Coord. Chem. Rev. 184, 319 – 346. Author, A., 1988. In: Lancaster, J. Jr. (Ed.), The Bioinorganic Chemistry of Nickel. VCH, New York. Landolph, J.R., 1999. Role of free radicals in metal-induced carcinogenesis. In: Sigel, H., Sigel, A. (Eds.), Metal Ions in Biological Systems, vol. 36. Marcel Dekker, New York, pp. 445 – 483. Lee, J.E., Ciccarelli, R.B., Wetterhahn Jennette, K.E., 1982. Solubilization of the carcinogen nickel subsulfide and Ist reaction with deoxynucleic acid and protein. Biochemistry 21, 771 – 778. Liang, Q., Ananias, D.C., Long, E.C., 1998. Ni(II)·Xaa-XaaHis induced DNA cleavage: deoxyribose modification by a common activated intermediate derived from KHSO5, MMPP, or H2O2. J. Am. Chem. Soc. 120, 248 – 257. Liang, Q., Eason, P.D., Long, E.C., 1995. MetallopeptideDNA interactions: site-selectivity based on amino acid composition and chirality. J. Am. Chem. Soc. 117, 9625 – 9631. Liang, R., Senturker, S., Shi, X., Bal, W., Dizdaroglu, M., Kasprzak, K.S., 1999. Effect of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage. Carcinogenesis 20, 893 – 898. Lloyd, D.R., Phillips, D.H., Carmichael, P.L., 1997. Generation of putative intrastrand cross-links and strand breaks in DNA by transition metal ion-mediated oxygen radical attack. Chem. Res. Toxicol. 10, 393 – 400.
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Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J., 1997. Crystal structure of the nucleosome core particle at 2.8 A, resolution. Nature 389, 251 – 260. Lynn, S., Yew, F.H., Hwang, J.-W., Tseng, M.-J., Jan, K.Y., 1994. Glutathione can rescue the inhibitory effects of nickel on DNA ligation and repair synthesis. Carcinogenesis 15, 2811 – 2816. Mack, P., Dervan, P.B., 1990. Nickel-mediated, sequence-specific oxidative cleavage of DNA by a designed metalloprotein. J. Am. Chem. Soc. 112, 4604 – 4606. Masarwa, M., Cohen, H., Meyerstein, C., Hickman, D.L., Bakac, A., Espenson, J.H., 1988. Reactions of low-valent transition-metal complexes with hydrogen peroxide. Are +2 they ‘Fenton-like’ or not? 1. The case of Cu+ aq and Craq . J. Am. Chem. Soc. 110, 4293 –4297. McKay, D.J., Renaux, B.S., Dixon, G.H., 1986. Human sperm protamines: amino acid sequences of two forms of protamine P2. Eur. J. Biochem. 156, 5 –8. Meijler, M.M., Zelenko, O., Sigman, D.S., 1997. Chemical mechanism of DNA scission by (1,10-phenanthroline) copper. Carbonyl oxygen of 5-methylenefuranone is derived from water. J. Am. Chem. Soc. 119, 1135 –1136. Muller, J.G., Kayser, L.A., Paikoff, S.J., Duarte, V., Tang, N., Perez, R.J., Rokita, S.E., Burrows, C.J., 1999. Formation of DNA adducts using nickel(II) complexes of redox-active ligands: a comparison of salen and peptide complexes. Coord. Chem. Rev. 185-186, 761 –774. Mylonas, M., Malandrinos, G., Plakatouras, J., Hadjiliadis, N., Kasprzak, K.S., Kre˛ z; el, A., Bal, W., 2001. Stray Cu(II) may cause DNA and protein oxidative damage when coordinated to the C-terminal tail of histone H2A. Chem. Res. Toxicol. 14, 1177 – 1183. Nackerdien, Z., Kasprzak, K.S., Rao, G., Haliwell, B., Dizdaroglu, M., 1991. Nickel(II)- and cobalt(II)-dependent damage by hydrogen peroxide to the DNA bases in isolated human chromatin. Cancer Res. 51, 5837 –5842. Pogany, G.C., Corzett, M., Weston, S., Balhorn, R., 1981. DNA and protein content of mouse sperm. Exp. Cell Res. 136, 127 – 136. Rodriguez, H., Holmquist, G.P., D’Agostino, R. Jr., Keller, J., Akman, S.A., 1997. Metal ion-dependent hydrogen peroxide-induced DNA damage is more sequence specific than metal specific. Cancer Res. 57, 2394 –2403. Salnikow, K., Gao, M., Voitkun, V., Huang, X., Costa, M., 1994. Altered oxidative stress responses in nickel-resistant mammalian cells. Cancer Res. 54, 6407 –6412.
Shi, X., Dalal, N.S., Kasprzak, K.S., 1993. Generation of free radicals in reactions of Ni(II) – thiol complexes with molecular oxygen and model lipid peroxides. J. Inorg. Biochem. 50, 211 – 225. Sigel, H., Martin, R.B., 1982. Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 82, 385 – 426. Sohal, R.S., Dubey, A., 1994. Mitochondrial oxidative damage, hydrogen peroxide release, and ageing. Free Radical Biol. Med. 16, 621 – 626. Spears, J.W., 1984. Nickel as a ‘newer trace element’ in the nutrition of domestic animals. J. Anim. Sci. 59, 823 – 835. Speetjens, J.K., Collins, R.A., Vincent, J.B., Woski, S.A., 1999. The nutritional supplement chromium(III) tris(picolinate) cleaves DNA. Chem. Res. Toxicol. 12, 483 – 487. Stearns, D., Wetterhahn, K.E., 1997. The mechanisms of metal carcinogenicity. Chromium(VI)-induced genotoxicity: direct and indirect pathways. In: Hadjiliadis, N. (Ed.), Cytotoxic, Mutagenic and Carcinogenic Potential of Heavy Metals Including Metals Related to Human Environment. NATO ASI (Ser. 2), vol. 26. Kluwer Academic, Dordrecht, pp. 107 – 121. Sugden, K.D., Stearns, D.M., 2000. The role of chromium(V) in the mechanism of chromate-induced oxidative DNA damage and cancer. J. Environ. Pathol. Toxicol. Oncol. 19, 215 – 230. Tkeshelashvili, L.K., Reid, T.M., McBride, T.J., Loeb, L.A., 1993. Nickel induces a signature mutation for oxygen free radical damage. Cancer Res. 53, 4172 – 4174. Tkeshelashvili, L.K., McBride, T.J., Spence, K., Loeb, L.A., 1991. Mutation spectrum of copper-induced DNA damage. J. Biol. Chem. 266, 6401 – 6406. Tsou, T.-C., Lin, R.-J., Yang, J.-L., 1997. Mutational spectrum induced by chromium(III) in shuttle vectors replicated in human cells: relationship to Cr(III) – DNA interactions. Chem. Res. Toxicol. 10, 962 – 970. Tullius, T.D., 1987. Chemical snapshots of DNA: using hydroxyl radical to study the structure of DNA and DNA – protein complexes. Trends Biochem. Sci. 12, 297 – 300. Vincent, J.B., 1999. Mechanisms of chromium action: lowmolecular-weight chromium-binding substance. J. Am. Coll. Nutr. 17, 548 – 555. Yamamoto, K., Kawanishi, S., 1989. Hydroxyl free radical is not the main active species in site-specific DNA damage induced by copper(II) ion and hydrogen peroxide. J. Biol. Chem. 264, 15435 – 15440.
Induction of oxidative DNA damage by carcinogenic metals Wojciech Bal a,b,*, Kazimierz S. Kasprzak c a
Faculty of Chemistry, Uni6ersity of Wroclaw, Ul. F. Joliot-Curie 14, 50 -383 Wroclaw, Poland b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland c Laboratory of Comparati6e Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD, USA
Abstract The metal ions carcinogenic to humans are As, Be, Cd, Cr and Ni, and the candidates also include Co, Cu, Fe and Pt. A range of molecular mechanisms was proposed for these metals, reflecting their diverse chemical properties. The oxidative concept in metal carcinogenesis proposes that some complexes of the above metals (Co, Cr, Cu, Fe, Ni) formed in vivo undergo redox cycling, yielding reactive oxygen species and/or high valence metal ions which oxidize DNA. Some of the products of oxidative DNA damage, including 8-oxoguanine and strand breaks, induce mutations, which may lead to neoplastic transformation. The establishment of metal-binding modes in the cell nucleus and of their reactivity is crucial for the understanding of molecular events in metal carcinogenesis. We have proposed the binding sites for Ni(II) and Cu(II) in core histones (H3, H2A) and sperm protamines (HP2) and, using molecular models, provided evidence for the generation of promutagenic oxidative DNA damage by the bound metals. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carcinogenesis; DNA; Histones; Metals; Oxidative damage; Oxygen radicals
1. Introduction The list of metals and metalloids whose compounds are definitely carcinogenic to humans includes (in alphabetical order) As, Be, Cd, Cr and Ni. There are more candidate elements emerging from current research, including Co, Cu, Fe and Pt (Kasprzak and Buzard, 2000). These elements are spread all over the periodic table, and, accordingly, their chemical properties are quite diverse. Therefore, a range of molecular mechanisms has been proposed for these metals. * Corresponding author. Tel.: + 48-71-3757281; fax: +4871-3282348. E-mail address: [email protected] (W. Bal).
The oxidative concept in metal carcinogenesis proposes that complexes formed by some of the above metals (Co, Cr, Cu, Fe, Ni) in vivo in the vicinity of DNA catalyze redox reactions, whose products oxidize DNA. In turn, some of the products of DNA-oxidative damage induce mutations, which may lead to neoplastic transformation. Such products include modified bases (primarily 8-oxoguanine), abasic sites (primarily depurinated), base adducts of carbon-centered radicals (including DNA-crosslinked proteins), and single and double breaks in the phospho-sugar backbone of DNA (Aust and Eveleigh, 1999). Two important distinctions have to be made within the list of oxidative carcinogenic metals. The first one is to separate chromium from the
0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 1 ) 0 0 4 8 3 - 0
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others. Its uniqueness stems from its strict assignment of carcinogenicity to one level of oxidation, namely +6 (which is not accessible to other carcinogenic metals), as opposed to the physiologically essential, although still enigmatic Cr(III) as part of the glucose tolerance factor (Vincent, 1999). The other type of distinction, between Ni and the tentative carcinogens Co, Cu and Fe, is physiological rather than chemical. These metals are essential microelements with multiple physiological functions, and thus with specific endogenous transport and control mechanisms. Ni has also been named essential for mammals (Spears, 1984). However, no specific physiological functions have ever been found for it, and the specific character of respective experiments leads to a speculation that the positive result reflects the requirements of the bacteria of the digestive tract of experimental animals rather than the animals themselves.
2. Molecular mechanisms of chromium carcinogenesis Because of its uniqueness, chromium will be discussed first. As mentioned above, this metal is carcinogenic when administered as anionic chromate or dichromate [Cr(VI)], and not as a Cr(III) species, because Cr(VI) anions are much more easily taken up by cells via physiological sulfate and phosphate transporters (Stearns and Wetterhahn, 1997). These anions cannot interact with DNA in vitro, because of mutual electrostatic repulsion. The intracellular Cr(VI) is reduced by a variety of factors, including glutathione (GSH), glutathione reductase, carbohydrates and ascorbic acid. It is paradoxical that the carcinogenicity of chromates is induced by cellular defense systems poised against the oxidative assault. Many not yet fully characterized short-lived reactive species formed in this way contain Cr(V) and Cr(IV). The chromate reductions can either be one-electron reactions, e.g. Cr(VI)Cr(V), Cr(V) Cr(IV), or two-electron reactions, e.g. Cr(VI) Cr(IV), Cr(V) Cr(III), depending on the reductant. Oxygen, carbon and sulfur-centered radicals, formed during the reduction, attack and damage DNA
and other cellular components. The DNA damage includes base oxidation, depurination (guanine) and DNA cross-links with proteins and radical products of cellular reductants (Kasprzak, 1996; Kasprzak and Buzard, 2000; Sugden and Stearns, 2000). The final product of chromate reduction, Cr(III), is also involved in the cross-link formation and, perhaps, in causing base substitution mutations, through non-redox mechanisms (Tsou et al., 1997). It seems that Cr(III) can also maintain redox activity in vivo through Cr(III)/Cr(V) (Dillon et al., 2000) or Cr(III)/Cr(II) redox pairs (Speetjens et al., 1999), thus re-activating the radical cascade. 3. Mechanisms of the formation of radical species The remaining metals (Co, Cu, Fe, Ni) in their physiologically relevant forms are predominantly, although not exclusively, one-electron reactants (Kasprzak, 1996; Landolph, 1999). The general mechanism of oxygen activation by such metal ions is provided by Fenton/Haber –Weiss and autoxidation reactions, in which molecular oxygen and its and H2O2 partially reduced forms superoxide O− 2 serve as substrates. Reactions (1) and (2) present the conversion of H2O2 into hydroxyl radical (OH) via the oxidation of a metal cation (note that the metal species serves as a reducing agent): Mn + + H2O2 M(n + 1) + + OH − + OH (Fenton reaction)
(1)
The redox cycling of the metal cation is maintained by reduction with O− 2 : (2) M(n + 1) + + O2− Mn + + O2 The balance of these two reactions is: H2O2 + O2− O2 + OH − + OH (Haber–Weiss reaction) (3) The only requirement for the above scheme of metal ion-catalyzed formation of hydroxyl radicals from unavoidable by-products of oxygen metabolism (Sohal and Dubey, 1994) is that a given metal ion has two neighboring oxidation states (Mn + and M(n + 1) + ) available at the same
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conditions. In the absence of chelators, the Fenton reaction is driven by Cu(I), Fe(II) and Co(II), but not by Ni(II) aqua ions. It is, however, unrealistic to expect unbound metal species under cellular conditions, in the presence of a plethora of proteins, and millimolar concentrations of such cellular components as glutathione, ascorbate, other carboxylic acids and nucleotides. Chelation, affecting the redox potentials of metal ions, modulates their ability to participate in the redox cycle. The oxidized form of the metal can also originate from auto-oxidation in the presence of molecular oxygen: Mn + +O2 M(n + 1) + +O2− M
n+
(4)
+O +2H+ M(n + 1) + +H2O2 − 2
(5)
The occurrence of the above reactions as well depends strongly on the presence or absence of specific chelators. Eqs. (2) and (5) demonstrate that, depending on the reaction conditions, O− 2 may be either the reductant or oxidant for a metal ion. Metal complexes can also be involved in twoelectron reactions, which yield strong oxidants alternative to OH, such as oxo and peroxo species, and singlet oxygen (in a decomposition process). Mn + +H2O2 [MO]n + +H2O n+
M
+H2O2 [MO2]
(n − 2) +
+2H
(6) +
(7)
It is often difficult to differentiate between the free and metal-bound oxygen radical species. Each case is specific and requires elaborate studies. For example, it has been established that metal-bound oxygen species are formed only in the reaction of Cu(I) aqua ion with H2O2 (Masarwa et al., 1988), while the EDTA complex of Fe(II) catalyzes H2O2 decomposition to free OH radicals (Tullius, 1987; Celender and Cech, 1990).
4. Oxidative chemistry of nickel(II) The ‘uncomplexed’ Ni(II) does not activate H2O2 (Cotelle et al., 1992; Shi et al., 1993) and is
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only weakly bound, and generally innocent to double-stranded DNA (Lee et al., 1982; Kasprzak et al., 1986). On the other hand, the effects of Ni(II) on DNA in vivo suggest Fenton-like behavior (Tkeshelashvili et al., 1993). In chemical studies on potential ligands that might facilitate nickel’s oxidative activity it has been established that only low-spin Ni(II) complexes (mainly of square-planar structure) can be oxidized to Ni(III) in water solution (Lancaster, 1988). Quite interestingly, this is a common feature of Ni(II) complexes with oligopeptides, even as simple as tetraglycine (Bossu et al., 1978). However, for most of the peptides, the appropriate complexes are formed at high, non-physiological pH values above 9 (Sigel and Martin, 1982). The relevant exclusion from this rule is provided by peptides having a His residue at position 3, which form low-spin, square-planar complexes at physiological pH (Kozłowski et al., 1999). The simplest of such peptides, Gly-Gly-His, has been studied, and was indeed found to activate not only hydrogen peroxide, but also the molecular oxygen in ambient air (Bossu and Margerum, 1976). The subsequent work has, however, demonstrated that this high reactivity is a consequence of the presence of a free terminal His carboxylate (Bal et al., 1994), which can also convey carbon radical reactions (Burrows et al., 1998). The peptides with the modified carboxylate function (tripeptide amides or longer peptides) require H2O2 or other oxidants (e.g. oxone). Nevertheless, Ni(II) complexes of Gly-Gly-His, its analogues with positions 1 and 2 modified (Xaa-Yaa-His in general) (Liang et al., 1995, 1998) and DNA binding proteins, extended N-terminally with the Gly-Gly-His sequence have gained interest as potential specific DNA cleavage agents in the presence of oxidants (Mack and Dervan, 1990; Harford et al., 1996). Another approach is to design non-peptidic macrocyclic Ni(II) complexes as DNA damaging agents (Muller et al., 1999). This approach has allowed studies of covalent adduct formation (alkylation) at nucleobases, because the macrocycles can be designed so that their oxidation potentials have values similar to those of the central ion, and thus the intermediate radical species can be localized on the ligand rather than on the metal ion.
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5. Formation of oxidizing species in Ni(II) interactions with histones The above studies, although providing important insights into the potential mechanisms of DNA damage by nickel species, and perhaps having future practical applications, still do not answer the question of actual ligands for Ni(II) in vivo, which might turn it into a Fenton-like agent. In search of such ligands we turned our interest to histones, as the most abundant proteins of cell nucleus (with formal concentrations of ca. 3 mM) (Bal et al., 1999). The initial studies demonstrated that nuclear proteins promote DNA oxidation by Ni(II) and H2O2 (Kasprzak and Bare, 1989; Nackerdien et al., 1991) and that nucleohistone markedly enhances 8-oxo-dG formation by the same system (Datta et al., 1993). Our analysis of sequences of histones, reviewed previously (Bal and Kasprzak, 1997; Bal et al., 2000a) and confirmed by the published three-dimensional structure of nucleosome (Luger et al., 1997) indicated only two potential binding sites for Ni(II), one in histone H3 ( –CAIH – motif in positions 110– 113), and another in histone H2A (– ESHH – motif in positions 121– 124). We studied them using oligopeptide models and partial protein assemblies. The study of the – CAIH – motif revealed the formation of a low-spin complex with the oligopeptide as well as with the histone core tetramer in vitro, and demonstrated that such a complex facilitates the 2%-deoxyguanosine oxidation in the presence of H2O2 (Bal et al., 1995, 1996, 1999). The binding constant obtained for the histone tetramer was relatively high (log K of 4.26 –5.26, depending on the extent of protein aggregation). However, auto-oxidation of the peptide complex to a disulfide was detected, which diminished the binding constant substantially. In the studies of Ni(II) binding to the oligopeptide derived from histone H2A, – TESHHK – , we established that the initial complex formed at physiological pH is high-spin, and, quite expectably, does not activate hydrogen peroxide (Bal et al., 1998, 2000b). The binding affinity of H2A for Ni(II) is comparable to that of H3 (Bal et al., 2000a). However, binding of Ni(II) to the –TESHHK – domain, studied using this hexapep-
tide, as well as the 34-peptide representing the whole C-terminal domain of H2A, and the whole histone H2A, resulted in the hydrolysis of the Glu-Ser peptide bond, with the formation of Ni(II) complexes with the – SHHK – motif of the cut-off peptides (i.e. SHHK-amide or SHHKAKGK, respectively). The Ni(II) complex thus formed demonstrated, not surprisingly, oxidative abilities in the presence of H2O2, cleaving the plasmid DNA, and oxidizing nucleobases. Oxidation of the peptide was also observed. Summarizing this line of research, we can conclude that our model peptide studies provided the— so far unique— mechanism of formation of a square-planar Ni(II) complex, derived from the core histone. Such a complex, capable of participating in Fenton chemistry, is a good candidate for an in vivo agent of DNA oxidative damage. At this juncture, our studies also indicated that cysteine peptides, such as –CAIH – from histone H3, might provide an alternative to His-3 ligands.
6. Metal –protamine interactions Protamines are highly basic peptides that substitute for histones in the sperm cell nuclei. Sperm DNA appears to be particularly susceptible to oxidative assault, because the oxidative metabolism is particularly active in the sperm, and yet there are no DNA repair mechanisms in the highly compacted sperm chromatin (Pogany et al., 1981). This disadvantage is compensated by the abundance of copper–zinc superoxide dismutase, an enzyme protecting from the superoxide radicals that leak from the mitochondria (Gu et al., 1995). The tentative relationship between the sperm and nickel carcinogenesis is provided by the concept of paternally transmitted transgenerational carcinogenesis, applied to the epidemiology of cancer incidence in the progeny of fathers occupationally exposed to heavy metals (Anderson et al., 1994; Buckley, 1994). Mammalian protamines belong to two families: P1 and P2. P1 is expressed in all species, while P2 in substantial amounts is present in just a few species, including human, horse and mouse (McKay et al., 1986). In these species, P2 is
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absolutely necessary for fertility (Belokopytova et al., 1993). It was therefore very interesting to find out that human P2 (HP2) is a His-3 peptide. We undertook the study of Ni(II) and Cu(II) binding to HP2 and the oxidative properties of resulting complexes. To facilitate the studies, we used the N-terminal 15-peptide of HP2 (HP21 – 15) as a model. The results (Bal et al., 1997a,b; Liang et al., 1999) illustrated the dual role of HP21 – 15 as a chelator: it quenched the DNA strand-breaking ability of Cu(II) in the presence of H2O2, but activated that of Ni(II). In contrast to Ni(II), the Cu(II)–H2O2 system without HP21-15 generates strand breaks and base oxidation in doublestranded DNA efficiently (Yamamoto and Kawanishi, 1989) (see below). The structural basis for the quenching was provided in a subsequent NMR study. It revealed a specific structure assumed by the HP21 – 15 peptide upon metal binding, with the Tyr-8 ring, placed in the vicinity of the metal site, serving as a potential suicide target for radical attack (Bal et al., 2000c). The likely explanation for the differences of properties between Ni(II) and Cu(II) complexes is a different nature of the reactive oxygen species produced by each metal: a metal-bound one for Cu(II), similar to that found for the Cu(II) aqua ion– DNA interaction (Yamamoto and Kawanishi, 1989), and a diffusible one for Ni(II). The role of HP2 as a protectant against adventitious copper, and at the same time as an activator of another metal, possibly responsible for nickel-related sperm damage, emerged from these studies.
7. DNA oxidation by metal complexes The protamine results lead to copper and other essential metals as potential mediators of oxidative DNA damage. Until recently, the essential metals, copper and iron, were not considered as agents of oxidative DNA damage in vivo, because they are under strict physiological control during uptake, transport and metabolism. However, there is an emerging view that this control may not be sufficient in certain pathological situations, such as inflammation, intoxication, or intense oxidative metabolism, which may damage mitochon-
59
dria and overwhelm the antioxidant defense systems. The essential metals might then become local (ultimate) carcinogens, as suggested for copper and hepatic hyperplasia (Eagon et al., 1999). The spectra of the mutational damage of free or chromatin DNA exposed to iron, copper and cobalt are similar to each other (and to nickel in chromatin). This finding supports the notion of a common underlying mechanism, namely hydroxyl radical-like attack (Tkeshelashvili et al., 1991, 1993; Rodriguez et al., 1997; Lloyd et al., 1997). Nonetheless, quantitative differences between individual agents have been reported, reflecting the diversity of specific mechanisms, e.g. proportion of base damage to strand breaks (apparently low for diffusible radicals and high for metal-bound oxidants) (Kennedy et al., 1997). In fact, the search for potential ligands in vivo for these metals is still just beginning. Basic facts on the reactivities of various model chelators in vitro and in vivo are being established. Like in the case of nickel, the mode of chelation is decisive for the activity, although most of the systems reported are active in vitro. For iron, the formation of Fe(II)/Fe(III) redox pair is beyond doubt. Positive examples supporting its DNA damaging activity include EDTA and NTA as ligands. These two are not very discriminate between Fe(II) and Fe(III). However, iron complexes with o-phenanthroline and deferoxamine, which strongly stabilize Fe(II), and with citric acid, stabilizing Fe(III), are inactive (Landolph, 1999), the latter in vivo rather than in vitro (Hartwig and Schlepegrell, 1995). For copper, the formation of Cu(I)/Cu(II) redox pair is usually invoked. This is certainly true for the interaction of copper with DNA in the absence of other bioligands (Yamamoto and Kawanishi, 1989; Drouin et al., 1996) and for some well-established chelators, such as ophenanthroline (Meijler et al., 1997). On the other hand, our recent research on the copper complexes of aminoglycoside antibiotics indicated that both Cu(I)/Cu(II) and Cu(II)/Cu(III) redox couples may be accessible for the same bioligand (Jez; owska-Bojczuk et al., 2001a,b). Finally, in a study of Cu(II) binding to the – TESHHK – model of histone H2A we found not only specific complex formation, but also strong oxidative ac-
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tivity in the presence of H2O2, which involved the Cu(II)/Cu(III) redox system (Mylonas et al., 2001). In contrast with the nickel study, the hydrolysis was not required for this oxidative activity. The latter work indicates that adventitious copper may also find docking sites in nuclear proteins, where it can exert site-specific damage.
8. Conclusion This brief and necessarily incomplete review of essential metals as potential carcinogenic DNA oxidants indicates that individual mechanisms are controlled by the local availability of specific ligands. We would also like to highlight the competition for metal ions between target proteins and/or DNA on one site and cellular low molecular weight ligands on the other. The establishment of tentative affinity constants for nickel complexes with histones allowed for calculations indicating that at high intracellular GSH levels, Ni(II) binding to histones may be negligibly low (Bal et al., 1999, 2000a). Formation of such complexes is possible at GSH concentrations lower than 1 mM, which correlates with the effects of Ni(II) seen in GSH-challenged cell cultures (Lynn et al., 1994; Salnikow et al., 1994). Such results will undoubtedly have to be taken into account in search for the mechanisms of oxidative DNA damage by carcinogenic metals.
Acknowledgements Dr W. Bal acknowledges the financial support from the Alexander von Humboldt Foundation.
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