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ToxSci Advance Access published online on May 28, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg126
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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Received February 10, 2003; accepted April 18, 2003
© 2003 Society of Toxicology

Neurotoxicology

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

David S. Baskin 1*, Hop Ngo 1, Vladimir V. Didenko 1

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

* To whom correspondence should be addressed. E-mail: dbaskin{at}tmh.tmc.edu.


  Abstract

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-250µM 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 2µM based on manual detection of fluorescent attached cells and at 1µM 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 250µM 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.

Key Words: thimerosal, active caspase-3, apoptosis, toxicity, neurons, fibroblasts, DNA breaks, membrane damage, DAPI .


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