Genotoxicity of inorganic mercury salts based on disturbed microtubule function

Abstract

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 micro M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 micro M, and no-effect-concentrations were between 0.001 and 0.005 micro M. Clearly enhanced MN rates were found at 0.1 micro M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37 degrees C was seen above the no-effect-concentration of 2 mM, with an IC(50) of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 micro M and reaching complete inhibition of motility at 30 micro M, whereas benzonitrile up to 200 micro M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 micro M. This points to the relevance of interactions with the cellular spindle apparatus.

Dynamic Effects of Hg2+-induced Changes in Cell Volume

“…8) as Hg 2+ entered the cell under hypotonic stress. Previous reports have shown that Hg 2+ disrupt both microtubules [48] and actin filaments [23]. Blebbing has been attributed to weakened adhesion between the bilayer and the cytoskeleton or disorganization of the cytoskeleton [49].…”

Does Inorganic Mercury Play a Role in Alzheimer's Disease? A Systematic Review and an Integrated Molecular Mechanism

“…In vitro studies produced the following results: Mercury interferes with polymerization of microtubules [122,123], increases secretion of both 1-40 and 1-42 forms of Aβ and promotes hyperphosphorylation of tau protein [9,[124][125][126][127], changes mitochondrial structure inducing a stress-response in astrocytes [128], and interferes with cell-maturation [129] or other aspects of cell functioning, such as DNA repair, glutathione level, or linkage and structure of microtubules [119,130,131]. Mercury disturbs the interaction between tubulin and GTP [132], and the chelator DMSA can reverse this process [133], while amalgam exposure is toxic for nerve cells in vitro [134].…”

Potent antileukemic action of naphthoquinoidal compounds: evidence for an intrinsic death mechanism based on oxidative stress and inhibition of DNA repair

“…This assay is a non-specific genotoxicity test that detects fixed mutations and permanent DNA damage. This test detects chromosomal aberrations as micronuclei in dividing versus non-dividing cells, 37 which serve as a good biomarker of exposure to genotoxins. The presence of a micronucleus represents the disruption of small amounts of non-condensed chromatin, and their increased frequency is evidence of prior structural chromosome damage or changes in chromosome number.…”