Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate.

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.

Acetyl-CoA metabolism in amprolium-evoked thiamine pyrophosphate deficits in cholinergic SN56 neuroblastoma cells.

Inhibition of pyruvate (PDHC) and ketoglutarate (KDHC) dehydrogenase complexes induced by thiamine pyrophosphate deficits is known cause of disturbances of cholinergic transmission in the brain, yielding clinical symptoms of cognitive, vegetative and motor deficits. However, particular alterations in distribution of key acetylcholine precursor, acetyl-CoA, in the cholinergic neuron compartment of thiamine pyrophosphate-deficient brain remain unknown. Therefore, the aim of our work was to find out how amprolium-induced thiamine pyrophosphate deficits (TD) affect distribution of acetyl-CoA in the compartment of pure cholinergic neuroblastoma SN56 cells originating from murine septum. Amprolium caused similar concentration-dependent decreases in thiamine pyrophosphate levels in nondifferentiated (NC) and differentiated (DC) cells cultured in low thiamine medium. In such conditions DC displayed significantly greater loss of viability than the NC ones, despite of lesser suppressions of PDHC activities and tetrazolium salt reduction rates in the former. On the other hand, intramitochondrial acetyl-CoA levels in DC were 73% lower than in NC, which explains their greater susceptibility to TD. Choline acetyltransferase activity and acetylcholine content in DC were two times higher than in NC. TD caused 50% decrease of cytoplasmic acetyl-CoA levels that correlated with losses of acetylcholine pool in DC but not in NC. These data indicate that particular sensitivity of DC to TD may result from relative shortage of acetyl-CoA due to its higher utilization in acetylcholine synthesis.

Structural effects of Zn(2+) on cell membranes and molecular models.

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1mM Zn and higher concentrations affect cell membrane and molecular models.

Phenotype-dependent susceptibility of cholinergic neuroblastoma cells to neurotoxic inputs.

A preferential loss of brain cholinergic neurons in the course of Alzheimer's disease and other encephalopathies is accompanied by a proportional impairment of acetyl-CoA synthesizing capacity in affected brains. Particular susceptibility of cholinergic neurons to neurodegeneration might results from insufficient supply of acetyl-CoA for energy production and acetylcholine synthesis in these conditions. Exposure of SN56 cholinergic neuroblastoma cells to dibutyryl cAMP and retinoic acid for 3 days caused their morphologic differentiation along with the increase in choline acetyltransferase activity, acetylcholine content and release, calcium content, and the expression of p75 neurotrophin receptors. Acetyl-CoA content correlated inversely with choline acetyltransferase activity in different lines of SN56 cells. In differentiated cells, aluminum (1 mM), amyloid beta(25-35) (0.001 mM), and sodium nitroprusside (1 mM), caused much greater decrease of pyruvate dehydrogenase and choline acetyltransferase activities and cell viability than in nondifferentiated ones. Aluminum (1 mM) aggravated suppressory effects of amyloid beta on choline acetyltransferase and pyruvate dehydrogenase activities and viability of differentiated cells. Similar additive inhibitory effects were observed upon combined exposure of differentiated cells to sodium nitroprusside and amyloid beta(25-35). None or much smaller suppressory effects of these neurotoxins were observed in nondifferentiated cells. Increase in the fraction of nonviable differentiated cells positively correlated with losses of choline acetyltransferase, pyruvate dehydrogenase activities, and cytoplasmic cytochrome c content in different neurotoxic conditions. These data indicate that highly differentiated cholinergic neurons may be more susceptible to aluminum and other neurotoxins than the nondifferentiated ones due to relative shortage of acetyl-CoA, increased content of Ca(2+), and expression of p75 receptors, yielding increase in cytoplasmic cytochrome c and subsequently grater rate of death of the former ones.