Thimerosal Induces DNA Breaks, Caspase-3 Activation, Membrane Damage, and Cell Death in Cultured Human Neurons and Fibroblasts.

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12773768&dopt=Abstract

 
Toxicol Sci. 2003 May 28 [Epub ahead of print]. Related Articles, Links
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Thimerosal Induces DNA Breaks, Caspase-3 Activation, Membrane Damage, and Cell Death in Cultured Human Neurons and Fibroblasts.

Baskin DS, Ngo H, Didenko VV.

Department of Neurosurgery, Baylor College of Medicine, 6560 Fannin Suite 944, Houston, Texas 77030; Veterans Affairs Medical Center, Houston, TX, 77030.

Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125nM-250 micro M concentrations of thimerosal for 45 minutes to 24 hours. 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells, and TUNEL to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 hours of incubation, thimerosal toxicity was observed at 2 micro M based on manual detection of fluorescent attached cells and at 1 micro M level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24-hour incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 hours of incubation with 250 micro M thimerosal. 6-hour incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage, and initiate caspase-3 dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

PMID: 12773768 [PubMed - as supplied by publisher]

 

 

 

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