Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt–tungsten carbide
Introduction
Assessment of genotoxicity can be performed at different steps of the interaction and of the effects of the mutagen on DNA. The DNA-binding capacity can be evaluated for chemical mutagens in terms of DNA adducts. The direct DNA-breaking capacity can be estimated either by alkaline elution, nick translation or alkaline single cell gel electrophoresis (comet assay); the latter, expressed in comet tail length and tail moment, was shown to be more sensitive than the former and allows a cell by cell approach 1, 2, 3, 4. Depending on the stage of the cell cycle, repair capacities, genetic background of the cells and the type of mutagen considered, only a fraction of the induced DNA damage will lead to fixed mutations. These chromosome and/or genome mutations can be quantified by determining, e.g. micronuclei in interphases or chromosome aberrations in metaphases.
In another paper of this special issue by Miller et al., the authors showed that the results obtained with the in vitro micronucleus (MN) test correlated very well with the frequencies of chromosome aberrations induced by clastogens in the same cells. For mechanistic reasons, it might also be interesting to investigate the correlation between the extent of DNA breakage measured in individual cells by the comet assay and the frequencies of micronuclei found in the same experimental conditions; this comparison will allow to estimate the amount of DNA breakage translated into chromosome and/or genome mutations. Therefore in this study, the genotoxic effects of cobalt metal powder (Co), tungsten carbide particles (WC) and a cobalt–tungsten carbide (WC-Co; hard metal) mixture were compared for both endpoints (MN test and comet assay).
Mutagenic and carcinogenic effects of cobalt compounds have been reviewed recently 5, 6. Since it was assumed that ionic cobalt forms represented the biologically active species, most studies were performed with soluble cobalt compounds. Cobalt(II) was mostly non-mutagenic in bacterial test systems; however, in mammalian cell cultures, Co(II) induced DNA strand breaks in different cell types as well as DNA protein cross-links. Furthermore, Co(II) is clastogenic, inducing chromosomal aberrations, micronuclei and sister chromatid exchanges; all of these effects were observed at basically non-cytotoxic concentrations (for review, see [6]). The formation of reactive oxygen species (e.g. hydroxyl radicals) has been proposed as one possible mechanism of metal genotoxicity in general (reviewed in [7]) and for cobalt in particular [8].
In contrast, little attention has been paid to cobalt metal and its alloys. Hard metal alloys (e.g. cobalt–tungsten carbide) have remarkable properties of hardness and are used for the manufacturing of different tools requiring high (heat) resistance such as diamond polishing tools [9]. Different forms of pulmonary diseases have been described among workers exposed to hard metal dust, including asthma, fibrosing alveolitis (hard metal disease) and lung cancer 10, 11, 12. While an asthmatic reaction can be induced by cobalt alone [13], epidemiological [14]and experimental data [15]strongly suggest that pulmonary fibrosis and lung cancer are resulting from an interaction between cobalt metal and tungsten carbide particles. Lison et al. [16]suggested that the mechanism of this interaction proceeds through the oxidation of cobalt metal catalysed at the surface of carbide particles, a reaction which results in the production of cobalt ions on the one hand, and hydroxyl radicals on the other (due to the reduction of dissolved oxygen).
Since hydroxyl radicals are extremely reactive species with DNA-damaging properties 17, 18it was our aim to compare the genotoxic potential of cobalt metal (Co) alone, tungsten carbide (WC) alone, and a mixture of cobalt–tungsten carbide (WC-Co) particles.
The study involved an in vitro exposure of isolated human leukocytes (lymphocytes+monocytes) to the metallic compounds described above, and was set up to elucidate the underlying genetic mechanisms of cobalt/cobalt–tungsten carbide induced genotoxicity/mutagenicity (see also [4]). Two different endpoints were considered to assess cytogenetic damage. The cytokinesis-blocked micronucleus (MN) test was used to quantify chromosome damage 19, 20, and the alkaline single cell gel electrophoresis (comet) assay was used to quantify initial DNA breakage [21].
The analysis of the genotoxic potential of Co, WC and WC-Co with the comet assay showed that the hard metal mixture (WC-Co) was able to produce more treatment-specific DNA breaks than Co alone, while no (dose-dependent) effect was observed by a WC treatment. Significant increases of micronuclei frequencies were observed in cytochalasin B blocked lymphocytes after exposure to Co and WC-Co. It was concluded that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC (i.e. in WC-Co alloys) and suggested that physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers.
In general, when both cytogenetic methods were considered, the micronucleus test seemed to be less sensitive to assess the DNA damaging potential of the compounds involved, which is expected since it detects chromosomal aberrations (chromosome/genome mutations) and not just repairable DNA breakage. Combination of the comet assay and the in vitro micronucleus test might therefore be recommended so as to understand the mechanisms underlying mutagenicity of genotoxicants and to assess the lowest effective dose.
Section snippets
Particles
The following powders were used: (1) extra fine cobalt metal obtained from a cobalt refinery (99.87% purity; median particle size (d50), 4 μm) called hereafter Co; (2) tungsten carbide (Johnson Matthey 625655, 99.5% purity, d50<1 μm), called hereafter WC; (3) hard metal powder prepared in the laboratory (6% of extra fine cobalt metal particles+94% tungsten carbide particles), called hereafter WC-Co. Since the aim of the study was also to compare Co with WC-Co, all experiments were designed to
DNA migration in human leukocytes exposed to Co, WC and WC-Co
Data on the mean tail length (TL) and mean tail moment (TM) for leukocytes treated in vitro for 15 min with Co, WC or WC-Co are presented in Table 1. Blood cells were obtained from the same donor and treated under the same experimental conditions as those used for the MN test. Significant increases in DNA migration among isolated leukocytes exposed to the metal compounds were observed. Although WC induced a significant DNA migration already at low concentrations, no dose-dependent increase was
Discussion
The micronucleus (MN) test has the advantage to detect in interphase both acentric chromosome fragments due to DNA breakage and chromosome loss resulting from chromatid/chromosome lagging in anaphase. If combined with the cytokinesis-block method, it moreover allows the assessment of cell toxicity/cell cycle delay by counting the binucleates and the selection of first mitotic cells after exposure to a mutagen (see this issue: Fenech; Kirsch-Volders et al.). In contrast to the scoring of
Acknowledgements
The authors wish to thank Ilse Goris for her excellent technical help and adequate scoring of the samples. This work was supported by the Belgian Federal Office for Scientific, Technical and Cultural affairs (O.S.T.C.).
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