or without the addition of peroxide (Nackerdien et al., 1991), suggesting reaction of DNA-bound Ni2+ directly with oxygen. 8- Oxo-2'-deoxyguanine was isolated from kidneys of rats treated with FeIII–NTA, but not FeCl3 (Umemura et al., 1990a,b); 8-oxo-2'- deoxyguanine is itself a mutagenic substance.
Metals can cause protein–DNA cross-links by forming metal bridges, but they can also form covalent cross-links — for example, when chromatin is incubated with FeII–EDTA and hydrogen peroxide (Lesko et al., 1982). Thymine–tyrosine cross-links were identified in chromatin treated with hydrogen peroxide in the pres- ence of Fe3+ or Cu2+. As noted for oxidative damage, chelation with EDTA or NTA increased thymine–tyrosine cross-links produced by Fe3+ and suppressed those forming in the presence of Cu2+
(Dizdaroglu, 1992). DNA cleavage also occurs. Strand breaks were produced in cultured cells by NiCl2, Ni3S2, and crystalline NiS, but not by amorphous NiS (Sunderman, 1989). Chromosome breaks and sister chromatid exchange have been observed in peripheral lympho- cytes of workers exposed to chromium and nickel compounds (Sunderman, 1989). NiII-mediated DNA cleavage with oxidants shows selectivity based on the ligand (Mack & Dervan, 1992;
Muller et al., 1992). Important factors are the availability of free coordination sites, Ni2+/Ni3+ redox potential, and charge of the com- plex (Muller et al., 1992). Depurination following metal exposure has been described. In a model system, chromate released guanine, whereas Cu2+ and Ni2+ released adenine (Schaaper et al., 1987).
There was also species dependence; whereas chromic acid or chromium(VI) trioxide (CrO3) caused a significant release of guanine, none was detected with CrCl3. The mechanism appears to involve coordination of the metal to the N-7 position of the purine, with subsequent scission of the glycosidic bond linking the purine to the sugar-phosphate backbone.
DNA repair mechanisms are necessary to maintain genome integrity. DNA is under continual insult from endogenously generated reactive oxygen species as well as exogenous toxicants, chemicals, and mechanical stresses. Therefore, mechanisms have evolved to repair DNA continuously or, alternatively, to eliminate cells, by apoptosis, in which DNA is irreversibly damaged. Both DNA repair and apoptosis are targets of toxicity. One of the main mechanisms for repairing damaged DNA is to excise the altered, oxidized, or cross-linked bases — so-called excision repair. The
reparative machinery responds both to the altered nucleotide and to the resulting conformational change in the DNA. Mismatch repair of replication errors and recombinational repair of cross-links and double strand breaks are also potential targets of toxic metals (Hartwig et al., 2002), but most information has been derived concerning excision repair.
Two types of excision repair can be distinguished, based on excision of the base or the nucleotide. Both types of excision repair are inhibited by low concentrations of Ni2+, Co2+, Cd2+, and As3+
(Hartwig et al., 2002). The involvement of the metal ions is com- plex. Base excision repair following damage from nitrosourea analogues is inhibited by arsenite (Li & Rossman, 1989). Cadmium and nickel inhibit base excision repair following photolytic damage (Dally & Hartwig, 1997). Ni2+ and Cd2+ can interfere with nucleotide excision repair by affecting the initial step of recognition of DNA damage (Hartmann & Hartwig, 1998), whereas Co2+ affects both incision and reparative polymerization (Kasten et al., 1997).
Arsenite impairs incision at lower concentrations and ligation at higher concentrations (Hartwig et al., 1997). Hartwig et al. (2002) have used a model of benzo[a]pyrene-induced DNA damage to study the effects of nickel species. Both NiCl2 and NiO inhibited removal of adducts in cultured cells, with NiO being slightly more effective. The metal species differences were rather subtle, however, and, as Hartwig et al. (2002) note, “do not provide an explanation for the marked differences in carcinogenic potencies between water- soluble and particulate nickel compounds”. Inhibition of DNA repair by Pb2+ and Cd2+ following injury to bacteria or cultured mammalian cells by various carcinogens, alkylating agents, and UV radiation has been reviewed (Hartwig, 1994). Some species differences are suggested — for example, between an inhibitory effect of lead(II) chloride (PbCl2) and a lack thereof with lead acetate on DNA repair in X-ray-irradiated HeLa cells.
Metals may also cause cancer through non-mutagenic, or epi- genetic, mechanisms (Klein & Costa, 1997) that alter the structure of DNA without changing the base sequence itself. Two major mechanisms are DNA methylation and heterochromatin formation.
Methylation primarily affects the C5 position of cytosine in 5'-CpG- 3' (and, to a lesser extent, in CpNpG) sequences that cluster in so- called CpG islands in regulatory regions of many genes.
Hypermethylation leads to gene silencing and regulates processes of differentiation and development and cell-specific gene expression (Cedar & Razin, 1990). Loss of normal methylation can reactivate the gene. Heterochromatin is composed of protein-rich regions of the chromosome that normally remain condensed and transcrip- tionally silent through the cell cycle and contain late-replicating DNA. Spreading of heterochromatin to adjacent chromosomal regions silences the gene in those regions. Histone deacetylation and chromatin-associated non-histone protein, HP-1, are involved in heterochromatin spreading, and the methylation status of the DNA may also be important (Klein & Costa, 1997).
Even in its carcinogenic species, nickel is a weak mutagen.
Costa and co-workers (reviewed in Klein & Costa, 1997) have produced several lines of evidence showing an epigenetic mech- anism. Carcinogenic nickel compounds were hypothesized to induce methylation silencing of an X-linked senescence gene, contributing to cell immortalization (Klein et al., 1991). In another model, DNA condensation and coordinate hypermethylation by carcinogenic nickel compounds silenced a mutagenic target sequence transgene (Lee et al., 1995).