Interaction of metal salts with cytoskeletal motor protein systems
Introduction
The classification of carcinogens, as well as of germ cell mutagens, is in a state of present discussion. In particular, in Germany the Senate Commission of the DFG for the Investigation of Health Hazards in the Work Area (MAK-Commission) has issued new recommendations to distinguish between 5 groups of proven and suspected carcinogens (Neumann et al., 1998). The new classification system introduced in Germany in 1998 has been the result of a continuing discussion over about 10 years (Bolt et al., 1988).
This new classification includes as Category 4 ‘Substances with carcinogenic potential for which genotoxicity plays no or at most a minor role. No significant contribution to human cancer risk is expected, provided that the MAK-value is observed.’ Moreover, the new Category 5 comprises ‘substances with carcinogenic and genotoxic potential, the potency of which is considered so low that, provided that the MAK-value is observed, no significant contribution to human cancer risk is to be expected.’
The distinction of these two new categories means that the classification of carcinogens, in future, should be based much more on mechanisms by which carcinogenic effects are elicited experimentally. In principle, the concept goes even further, as it should probably not only be distinguished between ‘genotoxic’ and ‘non-genotoxic’ carcinogens, but within the group of ‘genotoxic’ carcinogens between those characterised by ‘threshold’ and ‘non-threshold’ effects.
Quite a number of organic industrial chemicals and inorganic compounds display chromosomal effects, which lead them to be viewed as ‘genotoxic compounds’. Such chemicals may induce experimental tumours at high doses. Non-threshold principles are still applied in defining permissible exposure values by most regulatory boards. Examples for these compounds include a wide variety of chemicals, such as carbonyl compounds (aldehydes, ketones), compounds with activated double bonds (e.g. allylic compounds), and chemicals strongly interacting with functional groups of proteins (e.g. heavy metal ions like Pb2+ and Hg2+).
Current research shows that an important target of macromolecular interaction of such compounds within target cells are cytoskeletal proteins (including tubulin, kinesin, dynein) which are involved in motor processes in eukaryotic cells, such as cell division in general and chromosomal segregation in particular. Especially, the motor functions of the spindle apparatus are affected by distinct proteins, i.e., tubulin and specific motor proteins (kinesin, dynein). The toxicological impact, the underlying mechanisms, and the dose–response characteristics of these macromolecular interactions represent in one of the mechanisms of ‘genotoxic’ response to chemicals, and are heretofore insufficiently investigated and understood.
This study aims at elucidating basic mechanisms of interactions of lead and mercury with cytoskeletal proteins as the key macromolecules of interaction with foreign chemicals leading to chromosomal genotoxic damage (e.g. aneuploidy, MN formation, etc.). It is supposed that for such interactions thresholds may be defined which could allow derivation of no-observed-adverse-effect-levels. This would be fundamental for setting health-based environmental and occupational standards in the future. Among inorganic chemicals, the effects of mercury and lead salts on functional activity of tubulin and kinesin were investigated by measurements of tubulin assembly and kinesin-driven motility and dose–response relationships determined. In addition, the cytotoxic and genotoxic potential was studied employing the neutral red assay and the MN test with CREST analysis.
Section snippets
MN assay and CREST analysis
Interactions of chemicals with cytoskeletal macromolecules are reflected by the micronucleus (MN) assay. Aneugenic compounds cause spindle or cinetochore damage and lead to the formation of MN containing complete chromosomes. Clastogens can induce structural chromosome breaks. Lead and mercury salts have been investigated regarding their ability to induce MN in V79 hamster fibroblasts. Distinction of aneugenic and clastogenic mechanisms was achieved with CREST analysis.
The MN assay was
MN assay and CREST analysis
Lead chloride and lead acetate induce, dose-dependently, MN in V79 cells. Fig. 1 presents the no-effect-concentration of lead chloride as 1.1 μM PbCl2 and of lead acetate as 0.05 μM Pb(II). The CREST analysis verified an aneugenic effect of Pb(II), which was already anticipated by MN size evaluation (data not shown).
Mercury chloride and mercury nitrate induced MN dose-dependently starting at the same concentration of 0.01 μM mercury(II). Maximal MN induction was seen at 0.1 μM (Fig. 2). CREST
Discussion
The concept underlying the present study is that genotoxicity as represented by aneugenic MN may be mediated through interactions with proteins of the cytoskeleton, i.e., with tubulin and/or the motor protein kinesin. These proteins are involved in the activity of the spindle apparatus of the cell and thereby in cell division. Such protein interactions should be characterised by conventional dose–response relationships, which may enable the definition of thresholds for genotoxicity of these
Acknowledgements
The studies are supported by CEFIC (CEFIC/LRI: CC-1FOAR-0003). Thanks are also due to C. Pütt for technical assistance.
References (16)
- et al.
Effect of microtubule-associated proteins on the protofilament number of microtubules assembled in vitro
Biochim. Biophys. Acta
(1984) - et al.
Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters
Cell Biol. Int.
(2000) - et al.
Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test
Mutat. Res.
(1992) - et al.
A comparison of the 8-hydroxydeoxyguanosine, chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes
Mutat. Res.
(1996) - et al.
Structural diversity and dynamics of microtubules and polymorphic tubulin assemblies
Electron Microsc. Rev.
(1990) - et al.
Cytogenetic study in workers occupationally exposed to mercury fulminate
Mutagenesis
(1991) - et al.
Stoffe mit begründetem Verdacht auf krebserzeugendes Potential (Abschnitt III, Gruppe B der MAK-Werte-Liste): Probleme und Lösungsmöglichkeiten
Arb. Sozialmed. Präven.
(1988) - DFG, 2002. Occupational Toxicants, vol. 17. Wiley-VCH, Weinheim, pp....
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