Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.
Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the 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 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (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 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of 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 h of incubation with 250 microM thimerosal. A 6-h 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.
Baskin
David S
DS
Department of Neurosurgery, Baylor College of Medicine, 6560 Fannin Suite 944, Houston, Texas 77030, USA. dbaskin@tmh.tmc.edu
Ngo
Hop
H
Didenko
Vladimir V
VV
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis*; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling; D007231:Infant, Newborn; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D012867:Skin / Q000166:cytology / Q000187:drug effects; D013849:Thimerosal / Q000633:toxicity*
D061945:Caspase Inhibitors; D011310:Preservatives, Pharmaceutical; D013849:Thimerosal; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases