Determining the oxidation states of manganese in NT2 cells and cultured astrocytes

Excessive brain manganese (Mn) can produce a syndrome called “manganism”, which correlates with loss of striatal dopamine and cell death in the striatum and globus pallidus. The prevalent hypothesis for the cause of this syndrome has been oxidation of cell components by the strong oxidizing agent, Mn³⁺, either formed by oxidation of intracellular Mn²⁺ or transported into the cell as Mn³⁺. We have recently used X-ray absorption near edge structure spectroscopy (XANES) to determine the oxidation states of manganese complexes in brain and liver mitochondria and in nerve growth factor (NGF)-induced and non-induced PC12 cells. No evidence was found for stabilization or accumulation of Mn³⁺ complexes because of oxidation of Mn²⁺ by reactive oxygen species in these tissues. Here we extend these studies of manganese oxidation state to cells of brain origin, human neuroteratocarcinoma (NT2) cells and primary cultures of rat astrocytes. Again we find no evidence for stabilization or accumulation of any Mn³⁺ complex derived from oxidation of Mn²⁺ under a range of conditions.     

Gain of function of Kir4.1 channel increases cell resistance to changes of potassium fluxes and cell volume evoked by ammonia and hypoosmotic stress

The Kir4.1 channel is an inward rectifying potassium channel involved in the control of potassium and water movement in mammalian cells. To evaluate independently the role of Kir4.1 alone and without interaction with other cellular effectors, we compared (86)Rb fluxes and cell volume in Kir4.1 transfected cells (Kir4.1(+)) with cells transfected with an empty vector (Kir4.1(-)). Transfection with Kir4.1 neither increased (86)Rb uptake nor (86)Rb efflux from cells in isotonic medium. Pretreatment with ammonia (5 mM ammonium chloride) in isotonic medium produced a pronounced increase of (86)Rb uptake and a moderate decrease of cell volume in Kir4.1(-) but not in Kir4.1(+) cells. However, pretreatment evoked no change in (86)Rb efflux in either cell type. Hypotonic treatment (HT) markedly increased (86)Rb efflux in Kir4.1(-) cells and increased cell volume in both cell types. Although pretreatment with ammonia did not alter the effect of HT on (86)Rb efflux in either Kir4.1(+) or Kir4.1(-) cells, it potentiated the effect of hypotonic treatment in increasing cell volume in Kir4.1(-) cells. The results demonstrate that the presence of Kir4.1 in cells increases their resistance to alterations of potassium fluxes and/or cell volume imposed by ammonia and hypotonicity.     

Glutathione modulation influences methyl mercury induced neurotoxicity in primary cell cultures of neurons and astrocytes

Methyl mercury (MeHg) is highly neurotoxic and may lead to numerous neurodegenerative disorders. In this study, we investigated the role of glutathione (GSH) and reactive oxygen species (ROS) in MeHg-induced neurotoxicity, using primary cell cultures of cerebellar neurons and astrocytes. To evaluate the effect of GSH on MeHg-induced cytotoxicity, ROS and GSH were measured using the fluorescent indicators chloro methyl derivative of di-chloro di-hydro fluorescein diacetate (CMH(2)DCFDA) and monochlorobimane (MCB). Cell-associated MeHg was measured with (14)C-radiolabeled MeHg. Mitochondrial dehydrogenase activity was detected by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. MTT timeline study was also performed to evaluate the effects of both the concentration and duration of MeHg exposure. The intracellular GSH content was modified by pretreatment with N-acetyl cysteine (NAC) or di-ethyl maleate (DEM) for 12 h. Treatment with 5 microM MeHg for 30 min led to significant (p<0.05) increase in ROS and reduction (p<0.001) in GSH content. Depletion of intracellular GSH by DEM further increased the generation of MeHg-induced ROS in both cell cultures. Conversely, NAC supplementation increased intracellular GSH and provided protection against MeHg-induced oxidative stress in both cell cultures. MTT studies also confirmed the efficacy of NAC supplementation in attenuating MeHg-induced cytotoxicity. The cell-associated MeHg was significantly (p<0.02) increased after DEM treatment. In summary, depletion of GSH increases MeHg accumulation and enhances MeHg-induced oxidative stress, and conversely, supplementation with GSH precursor protects against MeHg exposure in vitro.     

The Manganese Health Research Program (MHRP): status report and future research needs and directions

The manganese (Mn) research health program (MHRP) symposium was a full day session at the 22nd International Neurotoxicology Conference. Mn is a critical metal in many defense and defense-related private sector applications including steel making and fabrication, improved fuel efficiency, and welding, and a vital and large component in portable power sources (batteries). At the current time, there is much debate concerning the potential adverse health effects of the use of manganese in these and other applications. Due to the significant use of manganese by the Department of Defense, its contractors and its suppliers, the Manganese Health Research Program (MHRP) seeks to use the resources of the federal government, in tandem with manganese researchers, as well as those industries that are involved with manganese, to determine the exact health effects of manganese, as well as to devise proper safeguard measures for both public and private sector workers. Humans require manganese as an essential element; however, exposure to high levels of this metal is sometimes associated with adverse health effects, most notably within the central nervous system. Exposure scenarios vary extensively in relation to geographical location, urban versus rural environment, lifestyles, diet, and occupational setting. Furthermore, exposure may be brief or chronic, it may be to different types of manganese compounds (aerosols or salts of manganese with different physical and/or chemical properties), and it may occur at different life-stages (e.g., in utero, neonatal life, puberty, adult life, or senescence). These factors along with diverse genetic composition that imposes both a background and disease occurrence likely reflect on differential sensitivity of individuals to manganese exposure. Unraveling these complexities requires a multi-pronged research approach to address multiple questions about the role of manganese as an essential metal as well as its modulation of disease processes and dysfunction. A symposium on the Health Effects of Manganese (Mn) was held on Wednesday, September 14, 1005, to discuss advances in the understanding on role of Mn both in health and disease. The symposium was sponsored by the Manganese Health Research Program (MHRP). This summary provides background on the MHRP, identifies the speakers and topics discussed at the symposium, and identifies research needs and anticipated progress in understanding Mn health- and disease-related issues.     

The transport of manganese across the blood-brain barrier

The mammalian central nervous system (CNS) possesses a unique and specialized capillary adaptation, referred to as the blood-brain barrier (BBB). The BBB maintains an optimal neuronal microenvironment, regulating blood-tissue exchange of macromolecules and nutrients. The BBB is characterized by individual endothelial cells that are continuously linked by tight junctions, inhibiting the diffusion of macromolecules and solutes between adjacent endothelial cells. This review will focus on pertinent issues to BBB maintenance, and survey recent dogmas on the transport mechanisms for the essential metal, manganese, across this barrier. Specifically, putative carriers for manganese into and out of the brain will be discussed.     

Increased manganese uptake by primary astrocyte cultures with altered iron status is mediated primarily by divalent metal transporter

Neurotoxicity due to excessive brain manganese (Mn) accumulation can occur via occupational exposure to aerosols or dusts that contain extremely high levels (>1-5 mg Mn/m(3)) of Mn, or metabolic aberrations (decreased biliary excretion). Given the putative role of astrocytes in regulating the movement of metals across the blood-brain barrier, we sought to examine the relationship between iron (Fe) status and Mn transport in astrocytes. Furthermore, our study examined the effect of Fe status on astrocytic transferrin receptor (TfR) and divalent metal transporter (DMT-1) levels and their relationship to Mn uptake, as both have been implicated as putative Mn transporters. All experiments were carried out in primary astrocyte cultures derived from neonatal rats when the cells reached full confluency (about three weeks in culture). Astrocytes were incubated for 24h in astrocyte growth medium (AGM) containing 200 microM desferroxamine (ID), 500 microM ferrous sulfate (+Fe), or no compound (CN). After 24h, 5 min (54)Mn uptake was measured and protein was harvested from parallel culture plates for DMT-1 and TfR immunoblot analysis. Both iron deprivation (ID) and iron overload (+Fe) caused significant increases (p<0.05) in (54)Mn uptake in astrocytes. TfR levels were significantly increased (p<0.05) due to ID and decreased in astrocytes exposed to +Fe treatments. As expected, DMT-1 was increased due to Fe deprivation, but surprisingly, DMT-1 levels were also increased due to +Fe treatment, albeit not to the extent noted in ID. The decreased TfR associated with +Fe treatment and the increased DMT-1 levels suggest that DMT-1 is a likely putative transporter of Mn in astrocytes.     

Characteristics of manganese (Mn) transport in rat brain endothelial (RBE4) cells, an in vitro model of the blood-brain barrier

Manganese (Mn), an essential elemental nutrient, is known to be neurotoxic at high occupational levels. We examined the transport of Mn across a monolayer of rat brain endothelial cell (RBE4) to evaluate whether an electromotive permeability mechanism is responsible for Mn transport across the blood-brain barrier (BBB). The (54)Mn(2+) apparent permeability and flux showed significant temperature-, energy- and pH-dependence, as well as partial sodium-dependence. Additionally, iron (Fe)-rich and Fe-deficient media significantly increased the apparent permeability of (54)Mn(2+). Finally, Mn flux and permeability decreased when RBE4 cells were grown in astrocyte-conditioned media (ACM), compared to standard alpha-media. These data reinforce observations that transport of Mn across the BBB occurs in part through active transport process.     

Effects of manganese on thyroid hormone homeostasis: potential links

Manganese (Mn) is an essential trace nutrient that is potentially toxic at high levels of exposure. As a constituent of numerous enzymes and a cofactor, manganese plays an important role in a number of physiologic processes in mammals. The manganese-containing enzyme, manganese superoxide dismutase (Mn-SOD), is the principal antioxidant enzyme which neutralizes the toxic effects of reactive oxygen species. Other manganese-containing enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and glutamine synthetase. Environmental or occupational exposure to high levels of manganese can cause a neuropathy resembling idiopathic Parkinson's disease, commonly referred to as manganism. Manganism and Parkinson's disease are both characterized by motor deficits and damage to nuclei of the basal ganglia, particularly the substantia nigra, with altered dopamine (and its metabolites) contributing to these disorders. Dopamine, a major neurotransmitter plays a crucial role in the modulation of the cognitive function, working memory and/or attention of the prefrontal cortex and the hippocampus. Dopamine is also a known inhibitory modulator of thyroid stimulating hormone (TSH) secretion. The involvement of dopamine and dopaminergic receptors in neurodevelopment, as well as TSH modulation, led us to hypothesize that excessive manganese exposure may lead to adverse neurodevelopmental outcomes due to the disruption of thyroid homeostasis via the loss of dopaminergic control of TSH regulation of thyroid hormones. This disruption may alter thyroid hormone levels, resulting in some of the deficits associated with gestational exposure to manganese. While the effects of manganese in adult populations are relatively well documented, comprehensive data on its neurodevelopmental effects are sparse. Given the importance of this topic, we review the potential participation of thyroid hormone dyshomeostasis in the neurodevelopmental effects of manganese positing the hypotheses that manganese may directly or indirectly affect thyroid function by injuring the thyroid gland or dysregulating dopaminergic modulation of thyroid hormone synthesis.     

Down-regulation of early ionotrophic glutamate receptor subunit developmental expression as a mechanism for observed plasticity deficits following gestational exposure to benzo(a)pyrene

The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children [Perera FP, Rauh V, Whyatt RM, Tsai WY, Tang D, Diaz D, et al. Effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children. Environ Health Perspect 2006;114:1287-92]. The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-d-aspartate receptor (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150mug/kg BW B(a)P on gestation days 14-17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in down-regulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: (1) disposition to the hippocampus and cortex, (2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and (3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes. These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.