Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH).
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.
Humphrey
Michelle L
ML
Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704-9388, USA.
Cole
Marsha P
MP
Pendergrass
James C
JC
Kiningham
Kinsley K
KK
D000935:Antifungal Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000473:pathology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects*; D009447:Neuroblastoma / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D013849:Thimerosal / Q000032:analysis / Q000633:toxicity*
D000935:Antifungal Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D013849:Thimerosal; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C022616:thiazolyl blue