diagnose during the first few months of life, but they later become clear (WHO, 1990a).
cell (e.g. by ascorbate), its genotoxic activity is suppressed, because CrIII species poorly penetrate cell membranes. The genotoxic activity of CrVI also depends on its capacity to undergo an intracellular reduction by a variety of systems (ascorbate, glutathione, hydrogen peroxide, cysteine, cytochrome P450 reductases, mitochondrial enzymes). Following intracellular reduction, several reactive intermediates are produced, including CrV, CrIV, and CrIII, as well as oxygen radicals. These secondary forms have the capacity to react with macromolecules and cause DNA damage (breaks, cross-links, adducts, etc.) (Shi et al., 1999a) and affect the fidelity of DNA replication (Singh & Snow, 1998).
In an in vitro study comparing the capacity of several transi- tional metals to produce oxygen radicals in a Fenton reaction (Lloyd et al., 1998), CrIII was the most potent species, followed by CrVI, VIII, FeII, and CuII. CrVI has also been shown to induce in Jurkat cells (a human T cell leukaemia cell line) the expression of the nuclear transcription factor NF kappa-B, which is particularly sensitive to cellular perturbations caused by reactive radicals (Shi et al., 1999b).
In cultured lung cells, the genotoxic activity of the poorly soluble carcinogen lead chromate has been shown to be mediated by the extracellular dissolution of the particles and not their internali- zation (Xie et al., 2004).
In vivo, it has been shown that intratracheally administered CrVI, but not CrIII, induced the formation of DNA strand breaks, measured by the alkaline DNA unwinding assay, in peripheral lymphocytes of rats (Gao et al., 1992).
In humans, the importance of chromium speciation for inducing genotoxic effects is not very well documented. Studies in popu- lations occupationally exposed to chromium compounds have shown that electroplating workers exposed to hexavalent chromium had an increased rate of micronucleus formation (Vaglenov et al., 1999) and sister chromatid exchanges (Wu et al., 2001), both assessed in circulating lymphocytes. No change in the rate of chromosomal aberrations could be detected in lymphocytes of workers exposed to CrIII in a ferrochromium factory in Italy (Sbrana et al., 1990). No study comparing, for the same genotoxic end-point, populations exposed to equivalent levels of CrIII and CrVI is available.
Since CrIII compounds are essential nutrients required for proper insulin function and normal protein, fat, and carbohydrate metabo- lism, the potential toxicity of several bioavailable ligand species has been investigated. Chromium picolinate has been shown to be mutagenic, and the picolinic acid moiety appears to be in part responsible, as studies show that picolinic acid alone is clastogenic.
Niacin-bound CrIII has been demonstrated to be more bioavailable and efficacious as a nutrient, and no toxicity has been reported (Bagchi et al., 2002).
8.8.2 Cobalt
Data accumulated in recent years indicate that, depending on the cobalt species considered (Co2+ ions or cobalt metal), different Regarding genotoxicity, the production of reactive oxygen species by Co2+ ions and cobalt metal together with inhibition of DNA repair by Co2+ ions appear to be the predominant modes of action (Lison et al., 2001).
In vitro, soluble and insoluble cobalt compounds have shown evidence of genotoxicity. Karyotype analysis after exposure of peripheral blood cells to cobalt(II) chloride (CoCl2) led to the observation of aneuploidy. Poorly soluble cobalt(II) sulfide (CoS) was found to induce DNA strand breaks in Chinese hamster ovary cells (Robison et al., 1982). It was also shown that, in the presence of hydrogen peroxide, micromolar concentrations of Co2+ ions are able to mimic Fe2+ cations in a Fenton-like reaction and cause damage to DNA bases of human lymphocytes or isolated DNA through the production of hydroxyl radicals (Nackerdien et al., 1991; Kawanishi et al., 1994; Lloyd et al., 1997). DNA damage resulting from the generation of reactive oxygen species by CoCl2
and hydrogen peroxide was further investigated by Mao et al. (1996) with electron spin resonance, electrophoretic assays, and HPLC.
They showed that the oxidation potential of Co2+ can be modulated by chelators to alter its capacity to generate reactive oxygen species and DNA damage: anserine enhanced the reactivity of Co2+ ions, whereas 1,10-phenanthroline and deferoxamine reduced and sup- pressed it, respectively. Co2+ ions were also shown to substitute for zinc in protein–zinc finger domains, which control the transcription of several genes. This substitution may contribute to explain how Co2+ generates reactive oxygen species sufficiently close to DNA to toxicity outcomes can be observed (see also sections 8.4 and 8.9).
cause damage (Sarkar, 1995). In vitro, in human lymphocytes and purified DNA, in the absence of hydrogen peroxide, CoCl2 up to a concentration of 0.1 mmol/l did not induce DNA single strand breaks (Anard et al., 1997), supporting the idea that Co2+ ions damage DNA through a Fenton-like reaction. In contrast to Anard et al. (1997), however, DNA breakage was detected by De Boeck et al.
(1998) in human lymphocytes incubated with CoCl2.
Until 1997, no report was available on the genotoxicity of cobalt metal particles, and it was assumed to be similar to that of the solubilized species (i.e. the Co2+ ion). The current view is that cobalt metal possesses a biological activity independent of its ionic form.
Physicochemical studies have shown that cobalt metal, and not its Co2+ ionic species, is thermodynamically able to reduce oxygen to form reactive oxygen species. The kinetics of this process are, however, slow as a result of the poor oxygen binding capacity at the surface of cobalt metal particles (Lison et al., 1995). In this system, soluble Co2+ ions are produced during, but do not drive, the critical reaction — i.e. reactive oxygen species are not produced by a Fenton-like reaction as for Co2+ ions. Meanwhile, the same authors found that the capacity of cobalt particles to produce reactive oxygen species is markedly increased in the presence of tungsten carbide particles, such as in hard metal powders. Anard et al. (1997) demonstrated the capacity of cobalt metal to induce DNA breaks and alkali-labile sites in isolated DNA and human lymphocytes. This damage could be partially blocked by scavenging reactive oxygen species with formate, indicating the possible involvement of the hydroxyl radical. The same authors demonstrated that, when tested in a range of cobalt equivalent concentrations, a hard metal mixture consisting of 94% tungsten carbide and 6% cobalt particles induced significantly more (on average a 3-fold increase) DNA breaks and alkali-labile sites than cobalt particles alone, both in isolated DNA and in cultured human lymphocytes. Scavenging reactive oxygen species with formate completely prevented DNA damage, again consistent with the involvement of hydroxyl radicals in this effect.
Similar results were reported by De Boeck et al. (1998). The mechanism of this interaction is most probably associated with an enhanced capacity of the mixture to produce reactive oxygen species (see section 8.4).
A similarly greater genotoxicity of hard metal compared with cobalt particles was found with the cytokinesis-blocked micro- nucleus test applied on human lymphocytes (Van Goethem et al., 1997). The mechanism by which cobalt and tungsten carbide–cobalt induce micronuclei is not clearly identified; it might be the consequence of the direct clastogenic activity discussed above, but an aneugenic activity should not be overlooked, as centromere- positive micronuclei were detected after in vitro exposure to cobalt (De Boeck et al., 1998). De Boeck et al. (2003) subsequently demonstrated, again in cultured human lymphocytes, that the aneu- genic interaction between cobalt and carbide particles previously observed with tungsten carbide also applies to some other metallic carbides (chromium carbide [Cr3C2] and niobium(IV) carbide [NbC], but not molybdenum(II) carbide [Mo2C]).
In addition to the capacity of cobalt metal and Co2+ to indirectly cause DNA damage via the production of reactive oxygen species, Co2+ ions have been reported to affect DNA repair processes. In vitro, CoCl2 was shown to inhibit nucleotide excision repair after UV irradiation in human fibroblasts (Hartwig et al., 1991). CoCl2
inhibited the incision and polymerization steps of the DNA repair process in human fibroblasts treated with UV-C (Kasten et al., 1997). Since DNA damage repair is an essential mechanism of homeostasis maintenance, its inhibition may also account for a mutagenic or carcinogenic effect of Co2+. Competition with essential Mg2+ ions (Kasten et al., 1997) and binding to zinc finger domains in repair proteins were identified as potential modes of indirect genotoxic activity of Co2+ ions. It has also been reported that the DNA binding capacity of the p53 protein, which is also a zinc- dependent mechanism, can be modulated by Co2+ ions (Palecek et al., 1999; Meplan et al., 2000).
Overall, the available data indicate that an assessment of the genotoxicity of cobalt and its compounds requires a clear distinction between the different species of the element and needs to take into account the different mechanisms involved.