Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration‐dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μM lead chloride and 0.05 μM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato‐cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST‐positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral‐red uptake assay. In a cell‐free system, 20–60 μM lead salts inhibited tubulin assembly dose‐dependently. The no‐observed‐effect concentration of lead(II) in this assay was 10 μM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady‐state toward disassembly, e.g., by reducing the concentration of assembly‐competent tubulin dimers. The effects of lead salts on microtubule‐associated motor‐protein functions were studied using a kinesin‐gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel‐stabilized microtubules across a kinesin‐coated glass surface. There was a dose‐dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μM and reached half‐maximal inhibition of motility at about 50 μM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mol. Mutagen., 2005. © 2005 Wiley‐Liss, Inc.

[1]  D. Eastmond,et al.  A critical evaluation of centromeric labeling to distinguish micronuclei induced by chromosomal loss and breakage in vitro. , 1997, Mutation research.

[2]  I. Pratt,et al.  Regulatory recognition of indirect genotoxicity mechanisms in the European Union. , 2003, Toxicology letters.

[3]  A Elhajouji,et al.  Concepts of threshold in mutagenesis and carcinogenesis. , 2000, Mutation research.

[4]  J. Heddle,et al.  The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. , 1976, Mutation research.

[5]  P. Apostoli,et al.  Effects of four inorganic lead compounds on the proliferation and junctional coupling of cultured REL liver cells. , 2000, American journal of industrial medicine.

[6]  C Winder,et al.  The genotoxicity of lead. , 1993, Mutation research.

[7]  P. Jacquet,et al.  Toxicity, mutagenicity and teratogenicity of lead. , 1980, Mutation research.

[8]  K. Jamil,et al.  Genotoxic effect of lead nitrate on mice using SCGE (comet assay). , 2000, Toxicology.

[9]  M. Kirsch‐Volders,et al.  Elimination of micronucleated cells by apoptosis after treatment with inhibitors of microtubules. , 2002, Mutagenesis.

[10]  M. Valverde,et al.  Genotoxicity induced in CD-1 mice by inhaled lead: differential organ response. , 2002, Mutagenesis.

[11]  J. Błasiak,et al.  In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links. , 2003, Mutation research.

[12]  Jan G Hengstler,et al.  Carcinogenicity categorization of chemicals-new aspects to be considered in a European perspective. , 2004, Toxicology letters.

[13]  C. Cantor,et al.  Microtubule assembly in the absence of added nucleotides. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[14]  H. Bolt.  Genotoxicity--threshold or not? Introduction of cases of industrial chemicals. , 2003, Toxicology letters.

[15]  Luigi Perbellini,et al.  Lead induced DNA strand breaks in lymphocytes of exposed workers: role of reactive oxygen species and protein kinase C. , 2002, Mutation research.

[16]  Vladimir Gelfand,et al.  Bovine brain kinesin is a microtubule-activated ATPase. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Jagetia,et al.  Effect of various concentrations of lead nitrate on the induction of micronuclei in mouse bone marrow. , 1998, Mutation research.

[18]  M. Kirschner,et al.  A protein factor essential for microtubule assembly. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[19]  H Babich,et al.  Comparisons of two in vitro cytotoxicity assays-The neutral red (NR) and tetrazolium MTT tests. , 1988, Toxicology in vitro : an international journal published in association with BIBRA.

[20]  B. Miller,et al.  Application of antikinetochore antibody staining (CREST staining) to micronuclei in erythrocytes induced in vivo. , 1990, Mutagenesis.

[21]  Hwa Shik Youn,et al.  Lead disturbs microtubule organization in the root meristem of Zea mays , 2000 .

[22]  A. Hartwig.  Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review. , 1994, Environmental health perspectives.

[23]  R. Stracke,et al.  SPEEDING UP KINESIN‐DRIVEN MICROTUBULE GLIDING IN VITRO BY VARIATION OF COFACTOR COMPOSITION AND PHYSICOCHEMICAL PARAMETERS * , 2000, Cell biology international.

[24]  Micheline Kirsch-Volders,et al.  Indirect mechanisms of genotoxicity. , 2003, Toxicology letters.

[25]  H. Bolt,et al.  Interaction of metal salts with cytoskeletal motor protein systems. , 2003, Toxicology letters.

[26]  A Elhajouji,et al.  Indications for a threshold of chemically‐induced aneuploidy in vitro in human lymphocytes , 1995, Environmental and molecular mutagenesis.

[27]  C. Cantor,et al.  Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. , 1974, Journal of molecular biology.

[28]  T. Sofuni,et al.  Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test. , 1992, Mutation research.

[29]  F M Johnson,et al.  The genetic effects of environmental lead. , 1998, Mutation research.

[30]  K Wyszynska,et al.  Genotoxic effects of occupational exposure to lead and cadmium. , 2003, Mutation research.

[31]  A. Russo,et al.  The centromere as a target for the induction of chromosome damage in resting and proliferating mammalian cells: assessment of mitomycin C-induced genetic damage at kinetochores and centromeres by a micronucleus test in mouse splenocytes. , 1996, Mutagenesis.

[32]  K Steenland,et al.  Lead and cancer in humans: where are we now? , 2000, American journal of industrial medicine.

[33]  R. Marcos,et al.  Occupational exposure to lead and induction of genetic damage. , 2001, Environmental health perspectives.

[34]  R. Lin,et al.  Studies on cytotoxic and genotoxic effects of cadmium nitrate and lead nitrate in chinese hamster ovary cells , 1994, Environmental and molecular mutagenesis.

[35]  M. Fenech,et al.  The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. , 1993, Mutation research.

[36]  E. Aboul-ela.  The protective effect of calcium against genotoxicity of lead acetate administration on bone marrow and spermatocyte cells of mice in vivo. , 2002, Mutation research.