Interaction of metal salts with cytoskeletal motor protein systems

doi:10.1016/S0378-4274(02)00502-7

Toxicology Letters

Volumes 140–141, 11 April 2003, Pages 75-81

https://doi.org/10.1016/S0378-4274(02)00502-7 Get rights and content

Abstract

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl₂, 0.05 μM Pb(OAc)₂ and 0.01 μM HgCl₂. The in vitro results obtained for PbCl₂ correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295–298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO₃)₂ and 0.1 μM HgCl₂ in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

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.

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Interaction of metal salts with cytoskeletal motor protein systems

Abstract

Introduction

Section snippets

MN assay and CREST analysis

MN assay and CREST analysis

Discussion

Acknowledgements

Biochim. Biophys. Acta

Cell Biol. Int.

Mutat. Res.

Mutat. Res.

Electron Microsc. Rev.

Cytogenetic study in workers occupationally exposed to mercury fulminate

Mutagenesis

Stoffe mit begründetem Verdacht auf krebserzeugendes Potential (Abschnitt III, Gruppe B der MAK-Werte-Liste): Probleme und Lösungsmöglichkeiten

Arb. Sozialmed. Präven.