Urbano et al: Hexavalent Chromium Exposure, Genomic Instability and Lung Cancer
Figure 2. Proposed structure for L–Cr(III)–DNA ternary complexes. Adapted from Zhitkovich, 2005.
Back in the nineties, Zhitkovich and collaborators showed, in an in vitro study, that cysteine-Cr(III)-DNA and glutathione-Cr(III)-DNA cross-links were the most abundant Cr(III)-DNA ternary complexes (Zhitkovich et al, 1995). As to Asc-Cr(III)-DNA cross-links, which appear to be the most mutagenic of all Cr-DNA adducts (Quievryn et al, 2003; Zhitkovich, 2005), their detection in cultured cells required restoration of physiological levels of Asc (Quievryn et al, 2002). This finding may, at least in part, explain the lower mutagenicity observed under low Asc concentrations as compared to that observed with physiological concentrations (Quievryn et al, 2006). Interestingly, although results from shuttle-vector experiments have shown that these ternary adducts inhibit replication in human cells (Quievryn et al, 2003), they demonstrated very little, if any, blocking potential in acellular systems that used purified polymerases, suggesting that the inhibitory action observed in vivo results from an indirect effect, rather than from direct interference of these adducts with replicative polymerases. Finally, these ternary complexes represent a major form of Cr(VI)-induced toxicity, as human cells unable to remove these lesions upon inactivation of nucleotide excision repair (NER) became much more sensitive to apoptosis and clonogenic lethality (Reynolds et al, 2004). Although less frequent, Cr(III)-DNA binary complex formation was also observed (Zhitkovich, 2005). Apparently, the binary complexes can be generated directly by reaction of Cr(III) with DNA or indirectly through binding of reactive Cr(IV) or Cr(V) species to DNA (Zhitkovich, 2005). Contrary to Cr-DNA ternary adducts, these binary adducts are only weakly mutagenic (Voitkun et al, 1998; Quievryn et al, 2003). DNA-Cr(III)-DNA interstrand cross-links (ICL) could only be detected during in vitro reduction of Cr(VI) by Asc (Bridgewater et al, 1994b; O’Brien et al, 2001; O’Brien et al, 2002) or cysteine (Zhitkovich et al, 2000), but not by glutathione (O’Brien et al, 2001). Also, their formation was revealed to be highly dependent on the ratio of reducer to Cr(VI), and the most extensive DNA crosslinking was always observed under conditions of limited reducer concentrations. Taking into account that the
formation of bifunctional complexes involving sterically hindered molecules, such as DNA, is an unlikely event, it was proposed that their formation would involved oligomeric Cr(III) complexes with each DNA strand bound to a different Cr(III) atom (Zhitkovich, 2005). Considering Cr(V)-DNA complexes, it has already been mentioned that Cr(V) formation is believed to occur only under very specific conditions. In any case, Cr(V) complexes exhibited little or no direct binding to DNA in vitro (Molyneux and Davies 1995; Levina et al, 2001) and were not required for the formation of Cr-DNA adducts (Quievryn et al, 2002, 2003; Reynolds et al, 2007; Reynolds and Zhitkovich, 2007). Moreover, in vitro studies on the formation of mutagenic damage showed that increased Cr(V) formation did not translate into higher levels of mutagenic damage (Quievryn et al, 2006). Also, Cr(V) was reported a weak mutagen in mammalian cells that rely on thiols to reduce Cr(VI) (Cohen et al, 1993). Altogether, these observations argue against an important role of Cr(V) in Cr(VI)-induced carcinogenicity. Cr(VI)-induced DNA lesions in mammalian cells also include the formation of cross-links between proteins and DNA (DPCs) (Zhitkovich, 1996a). Cr-DPCs are stable, ternary DNA adducts and constitute a significant class of Cr-related genetic lesions that may represent a major obstacle for the replication and transcription processes (Fornace et al, 1981; Manning et al, 1994; Wei et al, 2004; Schnekenburger et al, 2007). In both the liver and kidneys of injected rats, Cr-DPCs have been reported to extensively develop between DNA and non-histone proteins (Cupo and Wetterhahn, 1985a). Some of the proteins cross-linked to DNA were shown to be nuclear lamins, actin and nuclear matrix proteins (Miller and Costa, 1988; Miller et al, 1991). However, as much as 50% of the cross-linking did not involve a Cr atom but, instead, appeared to be catalyzed by oxidative mechanisms (Mattagajasingh and Misra, 1996). Although it was claimed that this type of lesion represents only a very small fraction of the initially formed DNA adducts in cultured cells, about 0.1% according to Zhitkovich (Zhitkovich, 2005), it is used as a biomarker of genetic damage in Cr-exposed human populations (Costa et al,
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