ABSTRACT
Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.
Subject(s)
Antifungal Agents/toxicity , Apoptosis/drug effects , Brain Neoplasms/pathology , Mitochondria/drug effects , Neuroblastoma/pathology , Thimerosal/toxicity , Blotting, Western , Caspase 3 , Caspase 9 , Caspases/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cytochromes c/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Microscopy, Fluorescence , Poly(ADP-ribose) Polymerases/metabolism , Subcellular Fractions/enzymology , Subcellular Fractions/metabolism , Tetrazolium Salts , Thiazoles , Thimerosal/analysisABSTRACT
Stem cells isolated from the central nervous system of both embryonic and adult mice can generate neurons and glia through the activation of different patterns of differentiation in dependence of exposure to appropriate epigenetic signals. On the other hand, environmental conditions might affect the proliferation, migration, and differentiation of these cells. We report here, for the first time, that inorganic mercury affects the proliferative and differentiative capacity of adult neuronal stem cells (ANSCs). Actually, inorganic mercury increases apoptosis in ASNC. Furthermore, in stem cell-derived astrocytes, high levels of the 70 kDa heat shock protein (HSP-70) occur, while the levels of GTP-beta-tubulin activity dramatically decrease. Interestingly, when induced to differentiate, inorganic mercury modifies morphological proprieties of astrocytes, while the neuron population is reduced. These results demonstrate that inorganic mercury produces toxicity in the ANSC-derived neuronal population and affects the biological properties of the glial-derived population.