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

• Published in: Toxicological sciences Vol. 74; no. 2; pp. 361 - 368
• Main Authors: Baskin, David S., Ngo, Hop, Didenko, Vladimir V.
• Format: Journal Article
• Language: English
• Published: Cary, NC Oxford University Press 01.08.2003
• Subjects: active caspase-3; apoptosis; Apoptosis - drug effects; Biological and medical sciences; Caspase 3; Caspase Inhibitors; Caspases - biosynthesis; Cell Membrane - drug effects; Cell Nucleus - drug effects; Cell Nucleus - pathology; Cell Survival - drug effects; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - drug effects; DAPI; DNA breaks; DNA Damage; Dose-Response Relationship, Drug; Drug toxicity and drugs side effects treatment; fibroblasts; Fibroblasts - drug effects; Fibroblasts - enzymology; Fibroblasts - pathology; Humans; In Situ Nick-End Labeling; Infant, Newborn; Male; Medical sciences; membrane damage; neurons; Neurons - drug effects; Neurons - enzymology; Neurons - pathology; Pharmacology. Drug treatments; Preservatives, Pharmaceutical - toxicity; Skin - cytology; Skin - drug effects; thimerosal; Thimerosal - toxicity; toxicity; Toxicity: nervous system and muscle; Toxicity; Cytotoxicity; membrane damage; neurons; Scission; Antifungal agent; fibroblasts; Enzymatic activity; Preservative; Cleavage; Mercury Organic compounds; DNA breaks; thimerosal; Human; Nervous system diseases; Enzyme; Caspase; DAPI; active caspase-3; In vitro; Peptidases; Neuron; Cell death; DNA; Hydrolases; Antibacterial agent; Fibroblast; Apoptosis
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfg126

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-μM 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 μM based on the manual detection of the fluorescent attached cells and at a 1-μM 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 μM 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.