Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding.

OBJECTIVES: Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. METHODS: Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. RESULTS: Gene expression analysis revealed that Ire1ß was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca(2+)-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. CONCLUSIONS: The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery.

N-acetylcysteine as a potential antidote and biomonitoring agent of methylmercury exposure.

BACKGROUND: Many people, by means of consumption of seafood or other anthropogenic sources, are exposed to levels of methylmercury (MeHg) that are generally considered to be quite low, but that may nevertheless produce irreversible brain damage, particularly in unborn babies. The only way to prevent or ameliorate MeHg toxicity is to enhance its elimination from the body. OBJECTIVES: Using N-acetylcysteine (NAC), we aimed to devise a monitoring protocol for early detection of acute exposure or relatively low MeHg levels in a rodent model, and to test whether NAC reduces MeHg levels in the developing embryo. RESULTS: NAC produced a transient, dose-dependent acceleration of urinary MeHg excretion in rats of both sexes. Approximately 5% of various MeHg doses was excreted in urine 2 hr after injection of 1 mmol/kg NAC. In pregnant rats, NAC markedly reduced the body burden of MeHg, particularly in target tissues such as brain, placenta, and fetus. In contrast, NAC had no significant effect on urinary MeHg excretion in preweanling rats. CONCLUSIONS: Because NAC causes a transient increase in urinary excretion of MeHg that is proportional to the body burden, it is promising as a biomonitoring agent for MeHg in adult animals. In view of this and because NAC is effective at enhancing MeHg excretion when given either orally or intravenously, can decrease brain and fetal levels of MeHg, has minimal side effects, and is widely available in clinical settings, NAC should be evaluated as a potential antidote and biomonitoring agent in humans.

Some aspects of astroglial functions and aluminum implications for neurodegeneration.

The present decade had witnessed an unprecedented attention focused on glial cells as a result of their unusual physiological roles that are being unraveled. It is now known that, rather than being a mere supporter of neurons, astroglia are actively involved in their modulation. The aluminum hypothesis seems to have been laid to rest, probably due to contradictory epidemiological reports on it as a causative factor of neurodegenerative diseases. Surprisingly, newer scientific evidences continue to appear and recent findings have implicated astrocytes as the principal target of its toxic action. In view of the likely detrimental effects of the interaction between these two infamous partners in neuroscience on neurons and nervous system, we have reviewed some aspects of glia-neuron interaction and discussed the implications of aluminum-impaired astrocytic functions on neurodegeneration. Because sporadic causes still account for the majority of the neurodegenerative diseases of which Alzheimer's disease is the most prominent, it has been suggested that neurotoxicologists should not relent in screening for the environmental agents, such as aluminum, and that considerable attention should be given to glial cells in view of the likely implications of environmental toxicants on their never-imagined newly reported roles in the central nervous system (CNS).

Accumulation of aluminum by primary cultured astrocytes from aluminum amino acid complex and its apoptotic effect.

Aluminum salts or doses that are unlikely in the human system have been employed in toxicity studies and much attention had been focused on the secondary target (neurons) of its toxicity rather than the primary target (astroglia). In order to address these issues, we have investigated the uptake and apoptotic effects of aluminum amino acid complex on primary cultured astrocytes because these are fundamental in understanding the mechanism of aluminum neurotoxicity. Aluminum solubilized by various amino acids was differentially internalized by astrocytes (glycine>serine>>glutamine>>glutamate), but aluminum was not internalized from citrate complex following 24 h of exposure. Inhibition of glutamine synthetase, by methionine sulfoximine (MSO), enhanced the uptake of aluminum from various amino acid complexes within 8 h except from glutamine complex. Blockade of selective GLT-1 (EAAT2) and GlyT1, as well as nonspecific transporters, did not inhibit or had no effect on uptake of aluminum in complex with the corresponding amino acids. Ouabain also failed to inhibit uptake of aluminum complexed with glycine. Pulse exposure to aluminum glycinate in the absence or presence of MSO caused apoptosis in over 25% of primary cultured astrocytes, and apoptotic features such as chromatin condensation and fragmentation became evident as early as 3 days of culture in normal medium. Lower doses (as low as 0.0125 mM) also caused apoptosis. The present findings demonstrate that aluminum solubilized by amino acids, particularly glycine, could serve as better candidate for neurotoxicity studies. Citrate may be a chelator of aluminum rather than a candidate for its cellular uptake. Amino acid transporters may not participate in the uptake of aluminum solubilized by their substrates. Another pathway of aluminum internalization may be implicated in addition to passive diffusion but may not require energy in form of Na+/K+-ATPase. Impaired astrocyes' metabolism can aggravate their accumulation of aluminum and aluminum can compromise astrocytes via apoptosis. Thus, loss of astrocytic regulatory and supportive roles in the central nervous system (CNS) may be responsible for neurodegeneration observed in Alzheimer's disease.

(13)C heteronuclear NMR studies of the interaction of cultured neurons and astrocytes and aluminum blockade of the preferential release of citrate from astrocytes.

Citrate has been identified as a major tricarboxylic acid (TCA) cycle constituent preferentially released by astrocytes. We undertook the present study to examine further the nature of metabolic compartmentation in central nervous system tissues using (13)C-labeled glucose and to provide new information on the influence of aluminum on the metabolic interaction between neurons and astrocytes. Metabolites released into the culture medium from astrocytes and neuron-astrocyte coculture, as well as the perchloric acid extracts of the cells were analyzed using 2D (1)H and (13)C NMR spectroscopy. Astrocytes released citrate into the culture medium and the released citrate was consumed by neurons in coculture. Citrate release by astrocytes was blocked in the presence of aluminum, with progressive accumulation of citrate within the cells. We propose citrate supply is a more efficient energy source than lactate for neurons to produce ATP, especially in the hypoglycemic state on account of it being a direct component of the TCA cycle. Astrocytes may be the cellular compartment for aluminum accumulation as a citrate complex in the brain.

Accelerated urinary excretion of methylmercury following administration of its antidote N-acetylcysteine requires Mrp2/Abcc2, the apical multidrug resistance-associated protein.

N-Acetylcysteine (NAC) is a sulfhydryl-containing compound that produces a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned mice, but the molecular mechanism for this effect is poorly defined. MeHg readily binds to NAC to form the MeHg-NAC complex, and recent studies indicate that this complex is an excellent substrate for the basolateral organic anion transporter (Oat)-1, Oat1/Slc22a6, thus potentially explaining the uptake from blood into the renal tubular cells. The present study tested the hypothesis that intracellular MeHg is subsequently transported across the apical membrane of the cells into the tubular fluid as a MeHg-NAC complex using the multidrug resistance-associated protein-2 (Mrp2/Abcc2). NAC markedly stimulated urinary [(14)C]MeHg excretion in wild-type Wistar rats, and a second dose of NAC was as effective as the first dose in stimulating MeHg excretion. In contrast with the normal Wistar rats, NAC was much less effective at stimulating urinary MeHg excretion in the Mrp2-deficient (TR-) Wistar rats. The TR- rats excreted only approximately 30% of the MeHg excreted by the wild-type animals. To directly test whether MeHg-NAC is a substrate for Mrp2, studies were carried out in plasma membrane vesicles isolated from livers of TR- and control Wistar rats. Transport of MeHg-NAC was lower in vesicles prepared from TR- rats, whereas transport of MeHg-cysteine was similar in control and TR- rats. These results indicate that Mrp2 is involved in urinary MeHg excretion after NAC administration and suggest that the transported molecule is most likely the MeHg-NAC complex.

Heavy metal analysis of groundwater from Warri, Nigeria.

The levels of some ions of heavy metals known to be associated with petroleum industry operations, including Pb, Ni, V, Cr, Cd, Zn and Fe, were studied in untreated groundwater from Warri area, Nigeria by atomic absorption spectroscopy. Warri area is characterized by petroleum industry activities including a Refinery. With this in mind, the residential area was divided into Effurun junction, Waterside Ekpan village and the Refinery's vicinity. The concentrations of Pb, Ni and Fe measured (in mg x l(-1)) in the groundwater samples of all areas studied ranged from 0.06 to 0.44, 0.008 to 0.19 and 0.315 to 2.753 respectively, while V, Cr, Zn and Cd were present in very low concentrations, 0-0.85 x 10 (-3). The levels of Pb, Ni and Fe exceeded the threshold limits (0.01, 0.02 and 0.3 mg x l(-1), respectively) set by the WHO health-based guideline for drinking water and this could portend environmental hazards.