Abstract
This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 µM, and inhibition is complete at about 10 µM. In this range, the tubulin assembly is fully (up to 6 µM) or partially (~6–10 µM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 µM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a “gliding assay” mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 µM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 µM HgCl2. Between 15 and 20 µM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 µM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 µM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.
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Acknowledgements
The studies were supported by CEFIC (CEFIC/LRI: CC-1FOAR-0003). The dedicated stewardship by Dr. A. Sarrif is very much appreciated. The authors thank H. Wolfram for performing part of the in vitro assays. The Ph.D. thesis of D.B., submitted to the Faculty of Science of the Heinrich-Heine University, Düsseldorf, Germany, contains part of the data of the present publication.
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Bonacker, D., Stoiber, T., Wang, M. et al. Genotoxicity of inorganic mercury salts based on disturbed microtubule function. Arch Toxicol 78, 575–583 (2004). https://doi.org/10.1007/s00204-004-0578-8
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DOI: https://doi.org/10.1007/s00204-004-0578-8