RESUMO
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.
Assuntos
Antifúngicos/toxicidade , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Mitocôndrias/efeitos dos fármacos , Neuroblastoma/patologia , Timerosal/toxicidade , Western Blotting , Caspase 3 , Caspase 9 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Microscopia de Fluorescência , Poli(ADP-Ribose) Polimerases/metabolismo , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismo , Sais de Tetrazólio , Tiazóis , Timerosal/análiseRESUMO
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.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Mercúrio/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Camundongos , Ligação Proteica/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismo , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/metabolismoRESUMO
A liquid chromatographic method using electrochemical detection is presented for measuring the thiol WR-1065 12-(3-aminopropylamino) ethanethiol] and its symmetrical disulfide WR-33278 [NH2(CH2)3NHCH2CH2S]2. WR- 1065 is the active, radioprotective drug derived from the phosphorothioate pro-drug WR-2721 (amifostine). External standard curves for both compounds were linear over the range of 40-200 pmol injected (r2 = 0.999 and 0.996 for the thiol and disulfide, respectively). The detection and quantitation limits for WR-1065 were 9 and 18 pmol, respectively, whereas the corresponding values for WR-33278 were 30 and 59 pmol, respectively. Within- and between-day determinations of measurement Vision and accuracy for both compounds validated the suitability of this assay method.
Assuntos
Mercaptoetilaminas/análise , Cromatografia Líquida de Alta Pressão , EletroquímicaRESUMO
New strategies for the prevention of radiation injuries are currently being explored with the ultimate aim of developing globally radioprotective, nontoxic pharmacologics. The prophylactic treatments under review encompass such diverse pharmacologic classes as novel immunomodulators, nutritional antioxidants, and cytokines. An immunomodulator that shows promise is 5-androstenediol (AED), a well-tolerated, long-acting androstene steroid with broad-spectrum radioprotective attributes that include not only protection against acute tissue injury, but also reduced susceptibility to infectious agents, as well as reduced rates of neoplastic transformation. Other potentially useful radioprotectants currently under study include the nutraceutical vitamin E and analogs, a chemically-engineered cytokine, interleukin-1beta, and a sustained-release formulation of an aminothiol, amifostine. Results suggest that a new paradigm is evolving for the prophylaxes of radiation injuries, based on use of newly identified, nontoxic, broad-spectrum prophylactic agents whose protective action may be leveraged by subsequent postexposure use of cytokines with organ-specific reparative functions.