**7. Nickel**

36 Selected Topics in DNA Repair

AP site), thereby interfering with the normal control of cell growth and division. Moreover cadmium exposure inhibits and modifies some proteins of BER such as formamidopyrimidine glycosylase (Fpg): the substitution of a cysteine in the zinc finger localized in the C terminal of Fpg protein may inhibit the binding of the protein to DNA (O' Connor et al., 1993). With respect to nucleotide excision repair, cadmium interferes with the removal of thymine dimers after UV irradiation by inhibiting the first step of this repair pathway (Hartwig & Schwerdtle 2002; Fatur et al. 2003). Also both association and dissociation of essential NER proteins are disturbed in presence of cadmium. Because of decreased of XPC nuclear protein levels, a reduced XPC localization to UVC-induced DNA damage in cells was observed after incubation with a non cytotoxic concentration of CdCl2. Interestingly, the tumor suppressor protein p53 also contain a zinc binding domain, which is essential for DNA binding and p53 function in transcription mechanism. In this context, Meplan et al. (1999) demonstrated that cadmium chloride alters p53 conformation in MCF7 cells, inhibits its DNA binding and down regulates transcriptional activation of a reporter gene. As p53 has been shown to act as a transcription factor for two important NER genes XPC and P48 and cadmium induced p53 conformational change may also result in altered p53 NER downstream effects (Adimoolam & Ford 2002). Cadmium exposure inhibits the xeroderma pigmentosum A (XPA) protein. XPA contains a typical four-cysteine zinc finger, which is not directly involved in DNA binding of the protein. The DNA binding capacity of XPA is strongly reduced after intoxication with cadmium (Hartmann et al., 1998; Hartwig et al., 2002). Another aspect is that cadmium found in liver and kidney cortex is bound to metallothioneins (MT), small, cystein-rich metal-binding proteins which are considered to be protective from cadmium toxicity (Klaassen et al., 1999; Nordberg 2009; Chang et al., 2009). Nevertheless, Hartwig et al 2002 demonstrated that the inhibitory cadmium effect for fpg proteins were comparable independent of whether CdCl2 or MT-bound Cd(II) was applied. Thus, metal ions complexed to MT may still be available for toxic reactions. In a recent study Schwerdtle et al., (2010) compared genotoxic effects of particulate CdO and soluble CdCl2 in cultured human cells and reported that both cadmium compounds inhibited the nucleotide excision repair of benzo[a]pyrene diol epoxide-induced bulky DNA adducts and UVCinduced photolesions in a dose-dependent shape at non-cytotoxic concentrations. This agreement with the similar carcinogenic effects of both water-soluble and water insoluble cadmium compound indicates that Cd2+ is the most common species responsible for

indirect genotoxicity of the element (Oldiges et al., 1989).

Among the carcinogenic metal compounds, only chromium (VI) has been clearly defined mutagenic in bacterial and mammalian test systems and its carcinogenic activity is thought to be due to the induction of DNA damage generated by reactive intermediates arising in its intracellular reduction to chromium (III) (Klein, 1996). Cr(VI)-carcinogenesis may be initiated or promoted through several mechanistic processes including, the intracellular metabolic reduction of Cr(VI) producing chromium species capable of interacting with DNA to yield genotoxic and mutagenic effects, Cr(VI)-induced inflammatory/immunological responses, and alteration of survival signaling pathways. The intracellular reduction of Cr(VI) produces a broad spectrum of DNA lesions including binary DNA adducts, DNA interstrand crosslinks (ICLs), DNA–protein adducts, DNA double-strand breaks and

**6. Chromium** 

Epidemiological studies in exposed workers identified some species of nickel as carcinogenic for upper respiratory tract and lung (Polednak 1981; Roberts et al. 1984; Roberts et al. 1989). The carcinogenic potency depends largely on properties such as solubility and kind of salts, which influence its bioavailability. Water soluble nickel salts are taken up only slowly by cells, while particulate of nickel compounds are phagocytosed and, due to the low pH, gradually dissolved in lysosomes, yielding high concentrations of nickel ions in the nucleus (Costa et al., 2005). Using in vitro cells and animal models, nickel compounds have been found to generate various types of adverse effects, including chromosomal aberrations, DNA strand breaks, high reactive oxygen species production, impaired DNA repair, hypoxia-mimic stress, aberrant epigenetic changes, and signaling cascade activation (Lu et al., 2005). Nickel has been shown to interfere with the repair mechanisms involved in removing UV-, platinum-, mitomycin C, g-radiation- and benzo[a]pyrene-induced DNA damage (Dally et al., 1997; Hartmann et al., 1998; Schwerdtle et al., 2002). These comutagenic effects are explained by the inhibition of all major types of DNA repair processes. Potentially sensitive targets for the toxic action of nickel(II) are zinc finger structures present in several DNA repair enzymes, including the bacterial Fpg protein and the mammalian XPA protein, DNA ligase III and poly(ADP-ribose) polymerase (PARP). Some studies investigated the effects of nickel compounds on the repair of DNA and showed that both soluble and particulate nickel can inhibit repair of benzo[a]pyrene DNA adducts in human lung cells (Schwerdtle et al., 2002). Low doses of nickel chloride (1 μmol/L) inhibited repair of UV or N-Methyl-N-nitro-N'-nitrosoguanidine -induced DNA damage as indicated by accumulating strand breaks, and 1–5 μm nickel chloride inhibited the formamidopyrimidine-DNA glycosylase (Fpg), 3-methyladenine-DNA glycosylase II (Alk A) and endonuclease III (Endo III) enzymes involved in DNA excision repair (Wozniak and Blaziak, 2004). The mechanisms of this action may include interactions with a specific structure containing zinc or the –SH groups of repair proteins. Because nickel compounds, such as NiS, Ni3S2, NiO (black and green), and soluble NiCl2, have been shown to be active inducers of reactive oxygen species (ROS) in Chinese hamster ovary cells, the involvement

Interactions by Carcinogenic Metal Compounds with DNA Repair Processes 39

trichloride (SbCl3) and antimony potassium tartrate (C4H4KO7Sb) on the repair of DNA double strand breaks induced by -radiation. Antimony compounds inhibited repair of DNA double strand breaks in a dose dependent manner. Both in trichloride, 0.2 mM antimony significantly inhibited the rejoining of double strand breaks, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses (D0) for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D0, but the antimony potassium tartrate inhibited the repair mechanism at doses where most cells lost their proliferating ability. This relationship is consistent with the general tendency of their respective toxicity: trivalent antimony compounds are less toxic than trivalent arsenic compounds, but more toxic than bismuth compounds (Leonard & Gerber, 1996; Huang et al., 1998). Grosskopf et al., (2010) show that trivalent antimony interferes with proteins involved in nucleotide excision repair and partly impairs this pathway, pointing to an indirect mechanism in the genotoxicity of trivalent antimony. After irradiation of human lung carcinoma cells with UVC, a higher number of cyclobutane pyrimidine dimers (CPD) remained in the presence of SbCl3, whereas processing of the 6−4 photoproducts and benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts were not impaired. Nevertheless, cell viability was reduced more than in additive mode after combined treatment of SbCl3 with UVC as well as with BPDE. A decrease in gene expression and protein level of XPE was found and moreover, trivalent antimony was shown to interact with the zinc finger domain of XPA with concentration dependent release of zinc from peptide of this domain. Compared to the data on arsenite, antimony is more effective in zinc releasing from XPA, yielding 50% zinc release at 10 times lower concentration (Schwerdtle et al., 2003). Antimony might be able to interact with proteins involved in DNA repair, via their cysteine or histidine side chains. Complexes between antimony(III) and glutathione via sulphur binding site of the

The carcinogenic potential of cobalt and its compounds was evaluated in 1991 by the International Agency for Research on Cancer (1991, 2006), the Commision concluded that cobalt and its compounds are possibly carcinogenic to humans (group 2B). Also the Deutsche Research Foundation (DFG 2008) has classified cobalt among the carcinogens of Category 2. Production of active oxygen species and inhibition of DNA repair appear to be the predominant mechanism of action in cobalt genotoxicity (Lison et al., 2001). Specifically by nucleotide excise repair pathway, in fact cobalt inhibits the removal of UV-induced cyclobutane pyrimidine dimers in mammalian cells but did not inhibit DNA strand rejoining after X-irradiation (Hartwig et al., 1991). Furthermore, by applying the nucleoid sedimentation assay in HeLa cells, Snyder et al (1989) demonstrated that cobalt causes an accumulation of DNA strand breaks after UV irradiation, indicating an impairment of the polymerization and/or the ligation step of nucleotide excision repair. Kasten et al. (1992) provided further evidence that cobalt at low non-cytotoxic concentration, inhibits both the incision and polymerization step of nucleotide excision repair in human fibroblasts. De Boeck et al., (1998) assessed the interference of cobalt compounds with the repair of primarily-induced DNA damage and showed that cobalt was able to cause persistence of methylmethanesulphonate-induced DNA lesions by interference its repair. In particular, cobalt inhibited the Xeroderma pigmentosum group A (XPA) protein, a zinc finger protein

tripeptide have already been confirmed (Burford et al., 2005).

**8.2 Cobalt** 

of reactive oxygen species has been implicated in the inhibition of DNA repair (Lynn, 1997). Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UVirradiated cells. Lynn et al., (1997) showed that nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely repair the inhibition by nickel or H2O2 alone but only partially the inhibition by nickel when associated with H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. Schwerdtle et al., (2002) studied the effect of soluble and particulated nickel compounds on the formation and repair of stable benzo(a)pyrene DNA adducts in human lung cells. With respect to adduct formation, NiO, but not NiCl2, reduced the generation of DNA lesions by ~30%. Regarding their repair in the absence of nickel compounds most lesions were removed within 24h; nevertheless, between 20 and 35% of induced adducts remained longer than 48h after treatment; NiCl2 (100µM) led to ~80% residual repair capacity; after 500µM the repair was reduced to ~36%. Also, even at the completely non-cytotoxic concentration of 0.5 µg/cm2 NiO, lesion removal was reduced to ~35% of control and to 15% at 2.0 µg/cm2. Nevertheless, under the same experimental conditions, the extent of DNA strand breaks and oxidative DNA base modifications were increased only at highly cytotoxic concentrations of both compounds (Hartwig et al., 2002). Repair inhibition by nickel appears therefore to be independent from metal compounds, and the results do not provide an explanation for the marked differences in carcinogenic potencies between soluble and particulated nickel species. However when considering the carcinogenicity in human or in experimental animals the retentions times in the body have to be taken into account. Thus, analysis of nickel contents in rat lungs after inhalation of different nickel species, especially for NiO, an impaired clearance and up to 1000-fold higher and persistent nickel lung burdens have been shown when compared to water-soluble nickel sulphate (Dunnick et al., 1995). Therefore, exposure to particulate nickel compounds may give rise to continuous DNA repair impairment and thus the biological consequences may be far more severe. The overall data add further evidences that the inhibition of DNA repair processes is an important mechanism in nickel genotoxicity, especially, because these effects are observed at low, non-cytotoxic concentrations. Since oxidative DNA damage is continuously induced during aerobic metabolism, an impaired repair of these lesions might explain the carcinogenic action of nickel(II).

### **8. Interaction on DNA repair processes of metallic elements classified as a possible or probable human carcinogen**

#### **8.1 Antimony**

Trivalent antimony is a known genotoxic agent and it is classified as a possible human carcinogen by the International Agency for Research on Cancer (1989) and as an animal carcinogen by the Deutsche Research Foundation (DFG 2008). The chemico-toxicological characteristics of antimony are similar to those of arsenic: their trivalent species are responsible for toxicological properties, and they have carcinogenic potential. In contrast to arsenic, however, informations about the toxicity of antimony and its possible mechanisms are limited. Tkahashi et al., (2002) investigated the effects of antimony trichloride (SbCl3) and antimony potassium tartrate (C4H4KO7Sb) on the repair of DNA double strand breaks induced by -radiation. Antimony compounds inhibited repair of DNA double strand breaks in a dose dependent manner. Both in trichloride, 0.2 mM antimony significantly inhibited the rejoining of double strand breaks, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses (D0) for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D0, but the antimony potassium tartrate inhibited the repair mechanism at doses where most cells lost their proliferating ability. This relationship is consistent with the general tendency of their respective toxicity: trivalent antimony compounds are less toxic than trivalent arsenic compounds, but more toxic than bismuth compounds (Leonard & Gerber, 1996; Huang et al., 1998). Grosskopf et al., (2010) show that trivalent antimony interferes with proteins involved in nucleotide excision repair and partly impairs this pathway, pointing to an indirect mechanism in the genotoxicity of trivalent antimony. After irradiation of human lung carcinoma cells with UVC, a higher number of cyclobutane pyrimidine dimers (CPD) remained in the presence of SbCl3, whereas processing of the 6−4 photoproducts and benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts were not impaired. Nevertheless, cell viability was reduced more than in additive mode after combined treatment of SbCl3 with UVC as well as with BPDE. A decrease in gene expression and protein level of XPE was found and moreover, trivalent antimony was shown to interact with the zinc finger domain of XPA with concentration dependent release of zinc from peptide of this domain. Compared to the data on arsenite, antimony is more effective in zinc releasing from XPA, yielding 50% zinc release at 10 times lower concentration (Schwerdtle et al., 2003). Antimony might be able to interact with proteins involved in DNA repair, via their cysteine or histidine side chains. Complexes between antimony(III) and glutathione via sulphur binding site of the tripeptide have already been confirmed (Burford et al., 2005).

#### **8.2 Cobalt**

38 Selected Topics in DNA Repair

of reactive oxygen species has been implicated in the inhibition of DNA repair (Lynn, 1997). Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UVirradiated cells. Lynn et al., (1997) showed that nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely repair the inhibition by nickel or H2O2 alone but only partially the inhibition by nickel when associated with H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. Schwerdtle et al., (2002) studied the effect of soluble and particulated nickel compounds on the formation and repair of stable benzo(a)pyrene DNA adducts in human lung cells. With respect to adduct formation, NiO, but not NiCl2, reduced the generation of DNA lesions by ~30%. Regarding their repair in the absence of nickel compounds most lesions were removed within 24h; nevertheless, between 20 and 35% of induced adducts remained longer than 48h after treatment; NiCl2 (100µM) led to ~80% residual repair capacity; after 500µM the repair was reduced to ~36%. Also, even at the completely non-cytotoxic concentration of 0.5 µg/cm2 NiO, lesion removal was reduced to ~35% of control and to 15% at 2.0 µg/cm2. Nevertheless, under the same experimental conditions, the extent of DNA strand breaks and oxidative DNA base modifications were increased only at highly cytotoxic concentrations of both compounds (Hartwig et al., 2002). Repair inhibition by nickel appears therefore to be independent from metal compounds, and the results do not provide an explanation for the marked differences in carcinogenic potencies between soluble and particulated nickel species. However when considering the carcinogenicity in human or in experimental animals the retentions times in the body have to be taken into account. Thus, analysis of nickel contents in rat lungs after inhalation of different nickel species, especially for NiO, an impaired clearance and up to 1000-fold higher and persistent nickel lung burdens have been shown when compared to water-soluble nickel sulphate (Dunnick et al., 1995). Therefore, exposure to particulate nickel compounds may give rise to continuous DNA repair impairment and thus the biological consequences may be far more severe. The overall data add further evidences that the inhibition of DNA repair processes is an important mechanism in nickel genotoxicity, especially, because these effects are observed at low, non-cytotoxic concentrations. Since oxidative DNA damage is continuously induced during aerobic metabolism, an impaired repair of these lesions might

**8. Interaction on DNA repair processes of metallic elements classified as a** 

Trivalent antimony is a known genotoxic agent and it is classified as a possible human carcinogen by the International Agency for Research on Cancer (1989) and as an animal carcinogen by the Deutsche Research Foundation (DFG 2008). The chemico-toxicological characteristics of antimony are similar to those of arsenic: their trivalent species are responsible for toxicological properties, and they have carcinogenic potential. In contrast to arsenic, however, informations about the toxicity of antimony and its possible mechanisms are limited. Tkahashi et al., (2002) investigated the effects of antimony

explain the carcinogenic action of nickel(II).

**8.1 Antimony** 

**possible or probable human carcinogen** 

The carcinogenic potential of cobalt and its compounds was evaluated in 1991 by the International Agency for Research on Cancer (1991, 2006), the Commision concluded that cobalt and its compounds are possibly carcinogenic to humans (group 2B). Also the Deutsche Research Foundation (DFG 2008) has classified cobalt among the carcinogens of Category 2. Production of active oxygen species and inhibition of DNA repair appear to be the predominant mechanism of action in cobalt genotoxicity (Lison et al., 2001). Specifically by nucleotide excise repair pathway, in fact cobalt inhibits the removal of UV-induced cyclobutane pyrimidine dimers in mammalian cells but did not inhibit DNA strand rejoining after X-irradiation (Hartwig et al., 1991). Furthermore, by applying the nucleoid sedimentation assay in HeLa cells, Snyder et al (1989) demonstrated that cobalt causes an accumulation of DNA strand breaks after UV irradiation, indicating an impairment of the polymerization and/or the ligation step of nucleotide excision repair. Kasten et al. (1992) provided further evidence that cobalt at low non-cytotoxic concentration, inhibits both the incision and polymerization step of nucleotide excision repair in human fibroblasts. De Boeck et al., (1998) assessed the interference of cobalt compounds with the repair of primarily-induced DNA damage and showed that cobalt was able to cause persistence of methylmethanesulphonate-induced DNA lesions by interference its repair. In particular, cobalt inhibited the Xeroderma pigmentosum group A (XPA) protein, a zinc finger protein

Interactions by Carcinogenic Metal Compounds with DNA Repair Processes 41

The International Agency for Research on Cancer has classified vanadium pentoxide (V2O5) as a possible carcinogen (Group 2B) (2006) while the Deutsche Research Foundation included vanadium among the carcinogens of Category 2 (DFG, 2008). The genotoxicity of vanadium compounds is explained by mechanisms of induction of oxidative stress, inhibition of DNA repair and interference with the activity of protein phosphatases and kinases. Only few studies have been carried out about the genotoxic action of vanadium compounds; Ivancsits et al. (2002) tested the impact of vanadate(V) on DNA repair kinetics of UV and bleomycin treated human fibroblasts. They observed a significant increase of DNA migration in the alkaline comet assay accompanied by persistent double-stranded breaks after exposure to vanadate in combination with UV-light or bleomycin, as compared to vanadate treatment alone. This indicates that vanadate may act as an indirect genotoxic agent by converting repairable single-stranded breaks into non-repairable double-stranded breaks. This effect was confirmed by the strong differences between lymphocytes of workers exposed to vanadium pentoxide after bleomycin treatment and controls. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-

The carcinogenic action of some metallic elements includes different mechanism such as induction of oxidative stress, inhibition of DNA repair, activation of mitogenic signalling, and epigenetic modification of gene expression. Nevertheless, each metallic elements and also each metal species exert characteristic interactions, and even though similar cellular pathways are affected, the underlying mechanisms are quite different. A relevant factor in metal carcinogenesis is the bioavailability of different metal species and the capacity to penetrate the cell barrier. The DNA does not appear to be the primary binding site for carcinogenic metal ions. This suggests that an inhibition of DNA repair processes may be a predominant mechanism in metal-induced genotoxicity. In addition, most carcinogenic metal compounds have been shown to increase the cytotoxicity, mutagenicity, and clastogenicity in mammalian cells when combined with different types of DNA-damaging agents (UV-light and/or alkylating agents). For most metal compounds, interactions with proteins appear to be more relevant for carcinogenicity as compared to direct DNA damage, and several targets have been identified, such as DNA repair, tumor suppressor and signal transduction proteins. Since metal ions can bind in principle to many electron rich centers in proteins the existence of particularly metal-sensitive protein structures may be suggested. The zinc finger proteins have been identified as potential molecular targets for toxic metal compounds and are involved not only in DNA binding but also in protein-protein interactions. Thus, there is an increasing evidence for zinc binding as structures very sensitive for toxic metal compounds. Significant factors appear to be not only the physicochemical properties but also accessibility and the protein microenvironment. The efficient repair of DNA lesions induced by endogenous processes and by environmental factors are an important prerequisite to maintain DNA integrity; if repair is not efficient, cells may accumulate DNA damage, leading to increased probabilities of genes instability and alteration in cellular cycle control and thus to tumor formation. The study and elaboration of metallic elements carcinogenicity should be conducted in parallel with doseresponse studies in order to have a real idea of exposures especially when considering the

**8.4 Vanadium** 

**9. Conclusion** 

induced lesions was reduced (Erlich et al., 2008).

possibility of co-exposures to other carcinogenic organic.

involved in nucleotide excision repair (Asmuß et al. 2000) where it substituted for the zinc ion (Kopera et al. 2004). Cobalt at low, non-cytotoxic concentrations interferes with the incision step of UV-induced DNA repair, but the removal of lesions may not be uniformly affected (Kasten et al., 1997). This effect may be related to differences in processing these lesions. Regarding the effect of cobalt on the incision frequency, a potentially preferential inhibition of incisions at 6-4-photoproducts could be due to either the disruption of the highly effective damage recognition at the site of this lesion or to a enhanced inhibition of the global genome repair system, while the transcription-coupled repair is unaffected at low doses. In addition to the incision step, the polymerization is inhibited by cobalt as well, while the ligation of repair patches is not affected by this element. A possible mechanism of the interference of cobalt with DNA polymerases could be the competition with magnesium; in fact the inhibition of the polymerization step was completely reversed in the presence of magnesium ions (Kasten et al. 1992, 1997). Sirover and Loeb (1976) demonstrated a dosedependent reduction of the catalytic activity as well as the fidelity of isolated DNA polymerases from different organisms after substitution of magnesium ions by cobalt. Taken together, the data indicate that cobalt belongs to a group of metal compounds which enhance the genotoxicity of direct mutagens.

#### **8.3 Lead**

The toxicity of lead and its compounds is well known for many centuries for anaemia, effects on nervous system and developmental disorders above all. Nevertheless, during the last years potential carcinogenic effects have been focused, leading to the classification of inorganic lead compounds as "Probably carcinogenic to humans" (Group 2A) by IARC (1987; 2006) and in the Group 2 by the Deutsche Research Foundation (DFG 2008). Although inorganic lead compounds exhibit only a weak mutagenic potential, they show more pronounced co-mutagenic activities in combination with DNA alkylating and oxidizing agents (Roy & Rossman, 1992; Hartwig et al., 1994). These effects were due to an interference with DNA repair processes, following an accumulation of DNA strand breaks, as shown in human HeLa cells after UV irradiation. Lead enhanced the frequencies of UV-induced mutations and sister chromatid exchanges at very low, nontoxic concentrations. Mutations as well as DNA strand breaks occurred only after long-term treatment at doses much higher than cytotoxic ones (Roy & Rossman, 1992). Considering the base excision repair, lead has been shown to inhibit the apurinic/apyrimidinic endonuclease (APE1) in micromolar concentration range both in an isolated enzymic test and in cells leading to an accumulation of apurinic sites in DNA and to an increase in methyl methansulfonate-induced mutagenicity (McNeill et al. 2007). Current evidences suggest that inactivation of APE1 is mediated by an unique and specific interaction of metal with active site residues then disrupting the in magnesiumdependent catalytic reaction. Furthermore, lead interferes with the repair of DNA double strand breaks via interaction with the stress response pathway induced by a phosphoinositol-3-kinase (PIKK) related kinase (Gastaldo et al. 2007). Due to its high affinity for sulfhydryl groups, a mechanism for lead interaction with proteins could be the displacement of zinc from zinc binding structures. In support of this assumption, in cellfree systems lead has been shown to reduce DNA binding of transcription factors (TFIIIA) and Sp1 (Huang et al. 2004). No impact was however described on the zinc-containing DNA repair proteins Fpg or XPA (Asmuß et al. 2000).

#### **8.4 Vanadium**

40 Selected Topics in DNA Repair

involved in nucleotide excision repair (Asmuß et al. 2000) where it substituted for the zinc ion (Kopera et al. 2004). Cobalt at low, non-cytotoxic concentrations interferes with the incision step of UV-induced DNA repair, but the removal of lesions may not be uniformly affected (Kasten et al., 1997). This effect may be related to differences in processing these lesions. Regarding the effect of cobalt on the incision frequency, a potentially preferential inhibition of incisions at 6-4-photoproducts could be due to either the disruption of the highly effective damage recognition at the site of this lesion or to a enhanced inhibition of the global genome repair system, while the transcription-coupled repair is unaffected at low doses. In addition to the incision step, the polymerization is inhibited by cobalt as well, while the ligation of repair patches is not affected by this element. A possible mechanism of the interference of cobalt with DNA polymerases could be the competition with magnesium; in fact the inhibition of the polymerization step was completely reversed in the presence of magnesium ions (Kasten et al. 1992, 1997). Sirover and Loeb (1976) demonstrated a dosedependent reduction of the catalytic activity as well as the fidelity of isolated DNA polymerases from different organisms after substitution of magnesium ions by cobalt. Taken together, the data indicate that cobalt belongs to a group of metal compounds which

The toxicity of lead and its compounds is well known for many centuries for anaemia, effects on nervous system and developmental disorders above all. Nevertheless, during the last years potential carcinogenic effects have been focused, leading to the classification of inorganic lead compounds as "Probably carcinogenic to humans" (Group 2A) by IARC (1987; 2006) and in the Group 2 by the Deutsche Research Foundation (DFG 2008). Although inorganic lead compounds exhibit only a weak mutagenic potential, they show more pronounced co-mutagenic activities in combination with DNA alkylating and oxidizing agents (Roy & Rossman, 1992; Hartwig et al., 1994). These effects were due to an interference with DNA repair processes, following an accumulation of DNA strand breaks, as shown in human HeLa cells after UV irradiation. Lead enhanced the frequencies of UV-induced mutations and sister chromatid exchanges at very low, nontoxic concentrations. Mutations as well as DNA strand breaks occurred only after long-term treatment at doses much higher than cytotoxic ones (Roy & Rossman, 1992). Considering the base excision repair, lead has been shown to inhibit the apurinic/apyrimidinic endonuclease (APE1) in micromolar concentration range both in an isolated enzymic test and in cells leading to an accumulation of apurinic sites in DNA and to an increase in methyl methansulfonate-induced mutagenicity (McNeill et al. 2007). Current evidences suggest that inactivation of APE1 is mediated by an unique and specific interaction of metal with active site residues then disrupting the in magnesiumdependent catalytic reaction. Furthermore, lead interferes with the repair of DNA double strand breaks via interaction with the stress response pathway induced by a phosphoinositol-3-kinase (PIKK) related kinase (Gastaldo et al. 2007). Due to its high affinity for sulfhydryl groups, a mechanism for lead interaction with proteins could be the displacement of zinc from zinc binding structures. In support of this assumption, in cellfree systems lead has been shown to reduce DNA binding of transcription factors (TFIIIA) and Sp1 (Huang et al. 2004). No impact was however described on the zinc-containing

enhance the genotoxicity of direct mutagens.

DNA repair proteins Fpg or XPA (Asmuß et al. 2000).

**8.3 Lead** 

The International Agency for Research on Cancer has classified vanadium pentoxide (V2O5) as a possible carcinogen (Group 2B) (2006) while the Deutsche Research Foundation included vanadium among the carcinogens of Category 2 (DFG, 2008). The genotoxicity of vanadium compounds is explained by mechanisms of induction of oxidative stress, inhibition of DNA repair and interference with the activity of protein phosphatases and kinases. Only few studies have been carried out about the genotoxic action of vanadium compounds; Ivancsits et al. (2002) tested the impact of vanadate(V) on DNA repair kinetics of UV and bleomycin treated human fibroblasts. They observed a significant increase of DNA migration in the alkaline comet assay accompanied by persistent double-stranded breaks after exposure to vanadate in combination with UV-light or bleomycin, as compared to vanadate treatment alone. This indicates that vanadate may act as an indirect genotoxic agent by converting repairable single-stranded breaks into non-repairable double-stranded breaks. This effect was confirmed by the strong differences between lymphocytes of workers exposed to vanadium pentoxide after bleomycin treatment and controls. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycininduced lesions was reduced (Erlich et al., 2008).

#### **9. Conclusion**

The carcinogenic action of some metallic elements includes different mechanism such as induction of oxidative stress, inhibition of DNA repair, activation of mitogenic signalling, and epigenetic modification of gene expression. Nevertheless, each metallic elements and also each metal species exert characteristic interactions, and even though similar cellular pathways are affected, the underlying mechanisms are quite different. A relevant factor in metal carcinogenesis is the bioavailability of different metal species and the capacity to penetrate the cell barrier. The DNA does not appear to be the primary binding site for carcinogenic metal ions. This suggests that an inhibition of DNA repair processes may be a predominant mechanism in metal-induced genotoxicity. In addition, most carcinogenic metal compounds have been shown to increase the cytotoxicity, mutagenicity, and clastogenicity in mammalian cells when combined with different types of DNA-damaging agents (UV-light and/or alkylating agents). For most metal compounds, interactions with proteins appear to be more relevant for carcinogenicity as compared to direct DNA damage, and several targets have been identified, such as DNA repair, tumor suppressor and signal transduction proteins. Since metal ions can bind in principle to many electron rich centers in proteins the existence of particularly metal-sensitive protein structures may be suggested. The zinc finger proteins have been identified as potential molecular targets for toxic metal compounds and are involved not only in DNA binding but also in protein-protein interactions. Thus, there is an increasing evidence for zinc binding as structures very sensitive for toxic metal compounds. Significant factors appear to be not only the physicochemical properties but also accessibility and the protein microenvironment. The efficient repair of DNA lesions induced by endogenous processes and by environmental factors are an important prerequisite to maintain DNA integrity; if repair is not efficient, cells may accumulate DNA damage, leading to increased probabilities of genes instability and alteration in cellular cycle control and thus to tumor formation. The study and elaboration of metallic elements carcinogenicity should be conducted in parallel with doseresponse studies in order to have a real idea of exposures especially when considering the possibility of co-exposures to other carcinogenic organic.

Interactions by Carcinogenic Metal Compounds with DNA Repair Processes 43

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