Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17β-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor β1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor β1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F 2 -isoprostanes (F 2 -IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 μ M MeHg for 1 or 6 hours. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (ΔΨm), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that ΔΨ m is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 μ M. MeHg pretreatment (1, 5 and 10 μ M) for 30 min also inhibited the net uptake of glutamine ( 3 H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 μ M) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 μ M) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.Please send request for reprints to Michael Aschner, Ph.D.,

Glutamine (Gln) is a growth determinant in neoplastic tissues. We analysed by RT-PCR the expression of mRNAs coding for the human variants of Gln transporters: ASCT2 (system ASC), SNAT1 [ATA1] (system A), SNAT3 [SN1] and SNAT5 [SN2] (system N), in samples of human malignant gliomas WHO grades III/IV (anaplastic astrocytoma and glioblastoma), glioma-derived cell cultures, brain metastases from peripheral organs, and control brain tissue. SNAT3 mRNA showed a 3-5 times stronger expression in gliomas than in metastases or control tissue, and was virtually absent from glioma cultures. Native glioblastoma immunostained positively with anti-SNAT3 antibody. The expression of ASCT2 mRNA, but not SNAT5 or SNAT1 mRNAs, was increased in all neoplastic tissues studied. Hence, increased expression of SNAT3 is a marker of primary malignant gliomas in situ.

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High demand of neoplastic tissues for glutamine (Gln) is met by its active transport across cell membranes. Chronic treatment with acrylamide in rodents is associated with an increased incidence of neoplasms, including astrocytomas. In this study, 24‐h acrylamide treatment significantly increased the initial rate of l‐[G‐3H]glutamine uptake in astrocyte cultures derived from the acrylamide‐sensitive Fischer 344 rat, and this effect could be fully inhibited by histidine, a model substrate for the amino acid transport system N. RT‐PCR analysis revealed that acrylamide treatment caused a significant increase in the astrocytic expression of the mRNA coding for the major system N protein, SNAT3, which is specifically overexpressed in malignant gliomas in situ. The acrylamide‐induced upregulation of astrocytic Gln transport via system N is likely to affect Gln homeostasis in these cells and may be causally related to the increased astrocytoma incidence observed in Fischer 344 rats.