**4. Beryllium**

Beryllium does not directly damage the DNA but it can lead to morphological cell transformation and inhibition of DNA repair synthesis. However, the effects observed on DNA repair are not specific for beryllium since similar findings are reported for other metallic compounds. A possible hypothesis is that the mechanism of genotoxicity is unlikely to be a non-threshold mechanism. A practical threshold can be postulated for beryllium since both direct DNA repair enzyme inhibition or DNA/protein expressionmediated effects do definitely require more than one ion to inhibit all DNA repair enzyme molecules (Strupp, 2011a). Dylevoĭ (1990), using four strains of E. coli with different DNA repairing capacities, established that beryllium efficacy in the DNA repair test depended on pH of medium and ions concentration. The DNA of rat primary hepatocytes was treated by incubation with 2-acetylaminofluorene, a known DNA damaging agent, and co-incubated with beryllium metal extracts (Strupp, 2011b). They observed that, the DNA repair synthesis were reduced by co-incubation with beryllium metal extract. However, it should be noted that this effect was observed only when the concurrent DNA damage was massive (>80% cells in repair), while no effects were observed in cells with lower DNA damage. These findings deserve however further investigations about their relevance in vivo.

### **5. Cadmium**

34 Selected Topics in DNA Repair

polymerase β (Pol β) and AP endonuclease (APE1), in response to low but physiologically relevant doses of arsenic. Lung fibroblasts and keratinocytes were exposed to As(III), and mRNA, protein levels and BER activity were assessed. Both Pol β and APE1 mRNA exhibited significant dose-dependent down regulation at doses of As(III) above 1 μM. However, at lower doses Pol β mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol β was rate limiting. These changes in BER are related to the overall protective against sunlight UVinduced toxicity at low doses of As(III) while at high doses there is a synergistic toxicity action. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis. The primary function of APE1 in BER is to act as an endonuclease responsible for the excision of apurinic/apyrimidinic (AP) sites. However, APE1 is also a redox factor responsible for signal transduction in response to oxidative stress (Hsieh et al., 2001). Arsenic has the potential to affect both the endonuclease and the functions of APE1, through its increase in

ROS levels and inhibition of DNA repair (Hamadeh et al., 2002).

Fig. 2. Schematic outline of DNA repair inhibition by arsenite and its methylated

Beryllium does not directly damage the DNA but it can lead to morphological cell transformation and inhibition of DNA repair synthesis. However, the effects observed on DNA repair are not specific for beryllium since similar findings are reported for other metallic compounds. A possible hypothesis is that the mechanism of genotoxicity is unlikely to be a non-threshold mechanism. A practical threshold can be postulated for beryllium since both direct DNA repair enzyme inhibition or DNA/protein expressionmediated effects do definitely require more than one ion to inhibit all DNA repair enzyme molecules (Strupp, 2011a). Dylevoĭ (1990), using four strains of E. coli with different DNA repairing capacities, established that beryllium efficacy in the DNA repair test depended

metabolites(modified from Hartwig et al., 2003).

**4. Beryllium** 

Several reports suggested that cadmium genotoxicity is not direct but rather mediated by reactive oxygen free radicals and resulting oxidative stress. In spite of being a weak genotoxic chemical, cadmium exhibits remarkable potential to inhibit DNA damage repair, and this has been identified as a major mechanism for its carcinogenicity (Giaginis et al., 2006). Cadmium is comutagenic and increases the mutagenicity of UV radiaton, alkylation and oxidation in mammalian cells. These effects may be explained by cadmium inhibition on several types of DNA repair: base excision repair, nucleotide excision repair, mismatch repair and the elimination of the premutagenic DNA precursor 8-oxodGTP. Regarding base excision repair, low concentrations of cadmium which did not generate oxidative damage as such, inhibited the repair of oxidative DNA damage in mammalian cells (Dally & Hartwig 1997; Fatur et al. 2003). Exposure of human cells to sub-lethal concentrations of cadmium leads to a time and concentration dependent decrease in hOGG1 activity, i.e. of the main DNA glycosylase activity responsible for the initiation of the base excision repair of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. The study of Bravard et al. (2010) confirms that part of the inhibitory effect of low dose cadmium on the cellular 8-oxoguanine DNA glycosylase activity can be attributed to an already described reduced hOGG1 transcription (Youn et al., 2005). This moderate inhibitory effect of cadmium on hOGG1 mRNA levels cannot explain the dramatic decrease observed in the levels and activity of hOGG1 protein. Indeed, inhibition of the ectopically expressed hOGG1-GFP in cells exposed to the metal confirmed the post-transcriptional effect of cadmium on hOGG1 protein and activity levels. A different response of the second enzyme in the cellular BER pathway has been described. Bravard et al (2010) found that in vivo treatment of human cells with cadmium has no effect on the APE1 activity, suggesting that in their experimental conditions most cadmium is complexed within the cells and therefore the intracellular concentrations of free cadmium do not reach the levels required for the inhibition of APE1. These results, taken together with the indirect inhibition of hOGG1 by oxidation, support the hypothesis that the effects on the BER pathway are in the consequence of the cellular redox imbalance rather than the direct interaction with proteins. Candelas et al. (2010) showed that cadmium inhibits the repair of uracile (U) in DNA, resulting both from mis-incorporation and cytosine (C) deamination. These lesions, as those on AP sites, are common in any cell, and must constantly be repaired to avoid mutagenic events. The necessity to continuously repair these lesions is underscored by the high levels of expression of UNG2 and APE1 (Cappelli et al., 2001). This genotoxic consequence of cadmium exposure might participate in the deregulation of physiological cellular processes by altering the pattern of gene expression on the one hand (U), and increasing the mutation rate on the other hand (on

Interactions by Carcinogenic Metal Compounds with DNA Repair Processes 37

oxidized bases (Nickens et al., 2010). On the contrary the knowledge about the role of DNA repair system in this process is lacking. Several lesions generated by Cr(VI) reduction (i.e. oxidized bases) are substrates for base excision repair (BER). In BER, damaged (alkylated or oxidized) bases are recognized by specific DNA glycosylases and are excised, resulting in the formation of apurinic/ apyrimidinic (AP) sites. Interesting to note that chromium(VI) can be reduced in body fluids, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes. Infact chromium(VI), when introduced by the oral route, is efficiently detoxified up reduction by saliva and gastric juice and sequestration by intestinal bacteria (De Flora, 2000). Administration of up to 20 mg chromium (VI), either in drinking water or by gavage, failed to produce any effect in the mouse bone marrow micronucleus assay or in the rat hepatocyte DNA rapair assay (Mirsalis et al., 1996).The results of studies carried out by O'Brien et al (2002; 2005) suggested that NER functions is essential in the protection of cells from Cr(VI) lethality and for the removal of Cr(III)-DNA adducts. Brooks et al., (2008) suggest that NER and BER are required for Cr(VI) genomic instability and postulate that, in the absence of excision repair, DNA damage is directed an

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

error-free system of DNA repair or damage tolerance.

**7. Nickel** 

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).

### **6. Chromium**

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 oxidized bases (Nickens et al., 2010). On the contrary the knowledge about the role of DNA repair system in this process is lacking. Several lesions generated by Cr(VI) reduction (i.e. oxidized bases) are substrates for base excision repair (BER). In BER, damaged (alkylated or oxidized) bases are recognized by specific DNA glycosylases and are excised, resulting in the formation of apurinic/ apyrimidinic (AP) sites. Interesting to note that chromium(VI) can be reduced in body fluids, which results in its detoxification, due to the poor ability of chromium(III) to cross cell membranes. Infact chromium(VI), when introduced by the oral route, is efficiently detoxified up reduction by saliva and gastric juice and sequestration by intestinal bacteria (De Flora, 2000). Administration of up to 20 mg chromium (VI), either in drinking water or by gavage, failed to produce any effect in the mouse bone marrow micronucleus assay or in the rat hepatocyte DNA rapair assay (Mirsalis et al., 1996).The results of studies carried out by O'Brien et al (2002; 2005) suggested that NER functions is essential in the protection of cells from Cr(VI) lethality and for the removal of Cr(III)-DNA adducts. Brooks et al., (2008) suggest that NER and BER are required for Cr(VI) genomic instability and postulate that, in the absence of excision repair, DNA damage is directed an error-free system of DNA repair or damage tolerance.
