Acrylonitrile has Distinct Hormetic Effects on Acetyl-Cholinesterase Activity in Mouse Brain and Blood that are Modulated by Ethanol.

Acrylonitrile(AN) is a neurotoxin both in animals and humans, but its effects on acetylcholinesterase (AChE) activity remain controversial. This study aimed to determine the dose-response effects of AN on AChE activity and the modulatory role of ethanol pre-treatment. A total of 144 Kunming mice were randomly divided into 18 groups: nine groups received 5% ethanol in their drinking water, and the remaining nine groups received regular tap water. One week later, both the ethanol and tap water only groups were given an intraperitoneal injection of AN at the following doses: 0 (control), 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 mg AN/kg body weight. AChE activity was determined on whole blood and brain 24 h later. Blood AChE activity was higher in AN-injected mice than in controls at all doses. AChE activity in blood increased in a dose-dependent manner, peaking at 0.156 mg/kg, after which a gradual decrease ensued, displaying a ß-typed dose-response relationship. In contrast, brain AChE activity, following a single AN injection, was consistently lower than in control mice, and continued to fall up to a dose of 0.313 mg/kg, and thereafter increased gradually with higher doses. Mice receiving a 20 mg/kg dose of AN exhibited AChE brain activity indistinguishable from that of control mice, demonstrating a typical U-typed dose-response relationship. The activity of AChE in the blood and brain of the AN + ethanol-treated groups displayed a shift to the right, and the magnitude of the decrease in AChE activity induced by AN was attenuated relative to the AN-only group. These results suggest that AN affects AChE activity in both mouse blood and brain in a hormetic manner. Pretreatment with ethanol modifies the effect of AN on AChE, indicating that parent AN has a more prominent role than its metabolites in modulating enzyme activity.

Ammonia increases paracellular permeability of rat brain endothelial cells by a mechanism encompassing oxidative/nitrosative stress and activation of matrix metalloproteinases.

Ammonia is responsible for cerebral edema associated with acute liver failure, but the role of the vasogenic mechanism has been a matter of dispute. Here, we tested the hypothesis that ammonia induces changes in blood-brain barrier (BBB) permeability by a mechanism coupled to oxidative/nitrosative stress (ONS) evoked in the BBB-forming cerebral capillary endothelial cells. Treatment of a rat brain endothelial cell line with ammonia (5 mmol/L, 24 h) caused accumulation of ONS markers: reactive oxygen species, nitric oxide and peroxidation products of phospholipid-bound arachidonic acid, F2-isoprostanes. Concurrently, ammonia increased the activity of extracellular matrix metalloproteinases (MMP-2/MMP-9), increased cell permeability to fluorescein isothiocyanate-dextran (40 kDa), and increased the expression of y+LAT2, a transporter that mediates the uptake to the cells of the nitric oxide precursor, arginine. The increase of cell permeability was ameliorated upon co-treatment with a MMP inhibitor, SB-3CT and with an antioxidant, glutathione diethyl ester, which also reduced F2-isoprostanes. Ammonia-induced ONS was attenuated by cytoprotective agents l-ornithine, phenylbutyrate, and their conjugate l-ornithine phenylbutyrate, an ammonia-trapping drug used to treat hyperammonemia. The results support the concept that ONS and ONS-related activation of MMPs in cerebral capillary endothelial cells contribute to the alterations in BBB permeability and to the vasogenic component of cerebral edema associated with acute liver failure.

Protective effect of a novel peptide against methylmercury-induced toxicity in rat primary astrocytes.

Methylmercury (MeHg) is an environmental neurotoxicant associated with aberrant central nervous system (CNS) functions. In this study, we examined the protective effect of a novel anti-inflammatory and cytoprotective nonapeptide, termed IIIM1, against MeHg-induced toxicity in cultured rat neonatal primary astrocytes. Astrocytes were pretreated for 66 h with 5 µg/ml IIIM1 (4.95 µM) followed by 6 h exposure to MeHg (5 µM). MeHg significantly increased F(2)-isoprostane generation, a lipid peroxidation biomarker of oxidative injury and this effect was significantly reduced upon pre-treatment with IIIM1. The MeHg-induced increase in levels of prostaglandin E(2) (PGE(2)), biomarkers of inflammatory responses, was also decreased in the peptide-treated cells. Mass spectrometry analysis revealed no chemical or binding interaction between MeHg and IIIM1, indicating that intracellular cytoprotective mechanism of action accounts for the neuroprotection rather than direct intracellular neutralization of the neurotoxicant with the peptide. These findings point to therapeutic potential for IIIM1 in a plethora of conditions associated with reactive oxygen species (ROS) generation. The implication of these findings may prove beneficial in designing new treatment modalities that efficiently suppress neurotoxicity, triggered not only by MeHg, but also by other metals and environmental agents, as well as chronic disease conditions that inherently increase reactive radical production and inflammatory signaling.

Protective effects of ebselen (Ebs) and para-aminosalicylic acid (PAS) against manganese (Mn)-induced neurotoxicity.

Chronic, excessive exposure to manganese (Mn) may induce neurotoxicity and cause an irreversible brain disease, referred to as manganism. Efficacious therapies for the treatment of Mn are lacking, mandating the development of new interventions. The purpose of the present study was to investigate the efficacy of ebselen (Ebs) and para-aminosalicylic acid (PAS) in attenuating the neurotoxic effects of Mn in an in vivo rat model. Exposure biomarkers, inflammatory and oxidative stress biomarkers, as well as behavioral parameters were evaluated. Co-treatment with Mn plus Ebs or Mn plus PAS caused a significant decrease in blood and brain Mn concentrations (compared to rats treated with Mn alone), concomitant with reduced brain E2 prostaglandin (PGE2) and enhanced brain glutathione (GSH) levels, decreased serum prolactin (PRL) levels, and increased ambulation and rearing activities. Taken together, these results establish that both PAS and Ebs are efficacious in reducing Mn body burden, neuroinflammation, oxidative stress and locomotor activity impairments in a rat model of Mn-induced toxicity.

The inhibitory effect of manganese on acetylcholinesterase activity enhances oxidative stress and neuroinflammation in the rat brain.

BACKGROUND: Manganese (Mn) is a naturally occurring element and an essential nutrient for humans and animals. However, exposure to high levels of Mn may cause neurotoxic effects. The pathological mechanisms associated with Mn neurotoxicity are poorly understood, but several reports have established it is mediated, at least in part, by oxidative stress. OBJECTIVES: The present study was undertaken to test the hypothesis that a decrease in acetylcholinesterase (AChE) activity mediates Mn-induced neurotoxicity. METHODS: Groups of 6 rats received 4 or 8 intraperitoneal (i.p.) injections of 25mg MnCl(2)/kg/day, every 48 h. Twenty-four hours after the last injection, brain AChE activity and the levels of F(2)-isoprostanes (F(2)-IsoPs) and F(4)-neuroprostanes (F(4)-NPs) (biomarkers of oxidative stress), as well as prostaglandin E(2) (PGE(2)) (biomarker of neuroinflammation) were analyzed. RESULTS: The results showed that after either 4 or 8 Mn doses, brain AChE activity was significantly decreased (p<0.05), to 60 ± 16% and 55 ± 13% of control levels, respectively. Both treated groups exhibited clear signs of neurobehavioral toxicity, characterized by a significant (p<0.001) decrease in ambulation and rearings in open-field. Furthermore, Mn treatment caused a significant increase (p<0.05) in brain F(2)-IsoPs and PGE(2) levels, but only after 8 doses. In rats treated with 4 Mn doses, a significant increase (p<0.05) in brain F(4)-NPs levels was found. To evaluate cellular responses to oxidative stress, we assessed brain nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and Mn-superoxide dismutase (Mn-SOD, SOD2) protein expression levels. A significant increase in Mn-SOD protein expression (p<0.05) and a trend towards increased Nrf2 protein expression was noted in rat brains after 4 Mn doses vs. the control group, but the expression of these proteins was decreased after 8 Mn doses. Taken together, these results suggest that the inhibitory effect of Mn on AChE activity promotes increased levels of neuronal oxidative stress and neuroinflammatory biomarkers.

Measurement of isoprostanes as markers of oxidative stress.

Oxidative stress results from an imbalance between production of reactive oxygen and nitrogen species (ROS and RNS, respectively) and endogenous antioxidant defense mechanisms. Increased generation of ROS/RNS is implicated in the pathogenesis of a variety of human diseases, including neurodegenerative disease, atherosclerosis, cancer, and aging. However, measuring oxidative stress in biological systems is complex and requires accurate quantification of either free radicals or damaged biomolecules. One method to quantify oxidative injury is to measure lipid peroxidation. Lipids are readily attacked by free radicals, resulting in the formation of a number of peroxidation products. F2-isoprostanes (F2-IsoPs) are one group of these compounds and they are derived by the free radical peroxidation of arachidonic acid (AA). The F2-IsoPs, prostaglandine F2-like compounds, provide an accurate measure of oxidative stress both in vitro and in vivo. This protocol details current methodology used to quantify these molecules using gas chromatography-mass spectrometry (GC/MS).

Protective effects of antioxidants and anti-inflammatory agents against manganese-induced oxidative damage and neuronal injury.

Exposure to excessive manganese (Mn) levels leads to neurotoxicity, referred to as manganism, which resembles Parkinson's disease (PD). Manganism is caused by neuronal injury in both cortical and subcortical regions, particularly in the basal ganglia. The basis for the selective neurotoxicity of Mn is not yet fully understood. However, several studies suggest that oxidative damage and inflammatory processes play prominent roles in the degeneration of dopamine-containing neurons. In the present study, we assessed the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates and associated neuronal dysfunctions both in vitro and in vivo. Results from our in vitro study showed a significant (p<0.01) increase in biomarkers of oxidative damage, F(2)-isoprostanes (F(2)-IsoPs), as well as the depletion of ATP in primary rat cortical neurons following exposure to Mn (500 µM) for 2h. These effects were protected when neurons were pretreated for 30 min with 100 of an antioxidant, the hydrophilic vitamin E analog, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or an anti-inflammatory agent, indomethacin. Results from our in vivo study confirmed a significant increase in F(2)-IsoPs levels in conjunction with the progressive spine degeneration and dendritic damage of the striatal medium spiny neurons (MSNs) of mice exposed to Mn (100mg/kg, s.c.) 24h. Additionally, pretreatment with vitamin E (100mg/kg, i.p.) or ibuprofen (140 µg/ml in the drinking water for two weeks) attenuated the Mn-induced increase in cerebral F(2)-IsoPs? and protected the MSNs from dendritic atrophy and dendritic spine loss. Our findings suggest that the mediation of oxidative stress/mitochondrial dysfunction and the control of alterations in biomarkers of oxidative injury, neuroinflammation and synaptodendritic degeneration may provide an effective, multi-pronged therapeutic strategy for protecting dysfunctional dopaminergic transmission and slowing of the progression of Mn-induced neurodegenerative processes.

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid ß-induced neurotoxicity.

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-ß (Aß). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aß-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aß-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aß led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24h following the exposure. The Aß-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F(2)-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aß neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration.

Progression of neurodegeneration and morphologic changes in the brains of juvenile mice with selenoprotein P deleted.

Selenoprotein P (Sepp1) is an important protein involved in selenium (Se) transport and homeostasis. Severe neurologic dysfunction develops in Sepp1 null mice (Sepp1(-/-)) fed a selenium-deficient diet. Sepp1(-/-) mice fed a selenium-deficient diet have extensive degeneration of the brainstem and thalamus, and even when supplemented with selenium exhibit subtle learning deficits and altered basal synaptic transmission and short-term plasticity in the CA1 region of the hippocampus. The goal of this study was to delineate the regional progression of neurodegeneration in the brain, determine the extent of neuronal cell death, and evaluate neurite structural changes within the hippocampus of Sepp1(-/-) mice. Whole brain serial sections of wild-type and Sepp1(-/-) mice maintained on selenium-deficient or supplemented diets over the course of 12 days from weaning were evaluated with amino cupric silver neurodegeneration stain. The neurodegeneration was present in all regions upon weaning and progressed over 12 days in Sepp1(-/-) mice fed selenium-deficient diet, except in the medial forebrain bundle and somatosensory cortex where the neurodegeneration developed post-weaning. The neurodegeneration was predominantly axonal, however the somatosensory cortex and lateral striatum showed silver-stained neurons. Morphologic analysis of the hippocampus revealed decreased dendritic length and spine density, suggesting that loss of Sepp1 also causes subtle changes in the brain that can contribute to functional deficits. These data illustrate that deletion of Sepp1, and presumably selenium deficiency in the brain, produce both neuronal and axonal degeneration as well as more moderate and potentially reversible neurite changes in the developing brain.

Prostanoid signaling: dual role for prostaglandin E2 in neurotoxicity.

The prostanoids, a naturally occurring subclass of eicosanoids, are lipid mediators generated through oxidative pathways from arachidonic acid. These cyclooxygenase metabolites, consisting of the prostaglandins (PG), prostacyclin and tromboxane, are released in response to a variety of physiological and pathological stimuli in almost all organs, including the brain. They are produced by various cell types and act upon targeted cells via specific G protein-coupled receptors. The existence of multiple receptors, cross-reactivity and coupling to different signal transduction pathways for each prostanoid, collectively establish their diverse effects. Notably, these effects can occur in functionally opposing directions within the same cell or organ. Prostaglandin E(2) (PGE(2)) is the most versatile prostanoid because of its receptors, E Prostanoid (EP) receptor subtypes 1 through 4, its biological heterogeneity and its differential expression on neuronal and glial cells throughout the central nervous system. Since PGE(2) plays an important role in processes associated with various neurological diseases, this review focuses on its dual neuroprotective and neurotoxic role in EP receptor subtype signaling pathways in different models of brain injury.

Methylmercury-induced alterations in astrocyte functions are attenuated by ebselen.

Methylmercury (MeHg) preferentially accumulates in glia of the central nervous system (CNS), but its toxic mechanisms have yet to be fully recognized. In the present study, we tested the hypothesis that MeHg induces neurotoxicity via oxidative stress mechanisms, and that these effects are attenuated by the antioxidant, ebselen. Rat neonatal primary cortical astrocytes were pretreated with or without 10 µM ebselen for 2h followed by MeHg (0, 1, 5, and 10 µM) treatments. MeHg-induced changes in astrocytic [(3)H]-glutamine uptake were assessed along with changes in mitochondrial membrane potential (ΔΨ(m)), using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Western blot analysis was used to detect MeHg-induced ERK (extracellular-signal related kinase) phosphorylation and caspase-3 activation. MeHg treatment significantly decreased (p<0.05) astrocytic [(3)H]-glutamine uptake at all time points and concentrations. Ebselen fully reversed MeHg's (1 µM) effect on [(3)H]-glutamine uptake at 1 min. At higher MeHg concentrations, ebselen partially reversed the MeHg-induced astrocytic inhibition of [(3)H]-glutamine uptake [at 1 min (5 and 10 µM) (p<0.05); 5 min (1, 5 and 10 µM) (p<0.05)]. MeHg treatment (1h) significantly (p<0.05) dissipated the ΔΨ(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. Ebselen fully reversed the effect of 1 µM MeHg treatment for 1h on astrocytic ΔΨ(m) and partially reversed the effect of 5 and 10 µM MeHg treatments for 1h on ΔΨ(m). In addition, ebselen inhibited MeHg-induced phosphorylation of ERK (p<0.05) and blocked MeHg-induced activation of caspase-3 (p<0.05-0.01). These results are consistent with the hypothesis that MeHg exerts its toxic effects via oxidative stress and that the phosphorylation of ERK and the dissipation of the astrocytic mitochondrial membrane potential are involved in MeHg toxicity. In addition, the protective effects elicited by ebselen reinforce the idea that organic selenocompounds represent promising strategies to counteract MeHg-induced neurotoxicity.

Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

γ-Glutamylcysteine ameliorates oxidative injury in neurons and astrocytes in vitro and increases brain glutathione in vivo.

γ-Glutamylcysteine (γ-GC) is an intermediate molecule of the glutathione (GSH) synthesis pathway. In the present study, we tested the hypothesis that γ-GC pretreatment in cultured astrocytes and neurons protects against hydrogen peroxide (H(2)O(2))-induced oxidative injury. We demonstrate that pretreatment with γ-GC increases the ratio of reduced:oxidized GSH levels in both neurons and astrocytes and increases total GSH levels in neurons. In addition, γ-GC pretreatment decreases isoprostane formation both in neurons and astrocytes, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation in astrocytes in response to H(2)O(2)-induced oxidative stress. Furthermore, GSH and isoprostane levels significantly correlate with increased neuron and astrocyte viability in cells pretreated with γ-GC. Finally, we demonstrate that administration of a single intravenous injection of γ-GC to mice significantly increases GSH levels in the brain, heart, lungs, liver, and in muscle tissues in vivo. These results support a potential therapeutic role for γ-GC in the reduction of oxidant stress-induced damage in tissues including the brain.

Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans.

Parkinson's disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn-induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration.

Peaceful use of disastrous neurotoxicants.

The increasing exposure to environmental neurotoxicants in the last decades caused serious health problems in the world population. Some of the neurotoxic agents are being used in agriculture and household such as insecticides and rodenticides and others are of natural origin like snake and scorpion venoms. Additional group of harmful substances is the chemical warfare agents including nerve and blistering agents that are known for their disastrous effects on neuronal tissues. The present paper presents a combination of epidemiological/clinical and molecular approaches for investigating the effect of certain groups of neurotoxicants on a variety of pathologies. The work of Finkelstein and coworkers describes epidemiological and clinical studies on acute and chronic organophosphate (OP)-induced neurotoxicity in certain populations in Israel. They mainly investigated the neurotoxic effects of low-level long-term exposure to OP in agricultural areas but also dealt with acute exposures as well. A molecular approach to OP mechanism of neuronal injury was described by Milatovic and coworkers. They demonstrated OP-induced oxidative injury in pyramidal neurons in the CA1 hippocampal area and its suppression by antioxidants. Lecht and coworkers described the novel snake venom angioneurins as important mediators of the physiological cross-talk between the cardiovascular and nervous systems. They also showed that under certain conditions these angioneurins may induce pathologies such as tumor development or disruption of the vascular barrier function during envenomation. Additional mechanistic/therapeutic approach was presented by Brodsky, Rosengarten, Proscura, Shapira and Wormser. They developed a novel anti-inflammatory peptide that reduced skin irritation induced by heat and sulfur mustard (SM) stimuli. Since SM causes neuropsychiatric symptoms and alterations in neurological functions this peptide may serve as a potential treatment of neuronal injuries caused by environmental neurotoxicants. These reviews highlight different aspects of neurotoxicity, addressing epidemiology and mechanisms of toxicity; and identifying novel potential therapies.

Mefloquine induces oxidative stress and neurodegeneration in primary rat cortical neurons.

Mefloquine is an effective antimalarial that can cause adverse neurological events including headache, nausea, fatigue, insomnia, anxiety and depression. In this study, we examined the oxidative stress response in primary rat cortical neurons treated with mefloquine by quantifying oxidative stress markers glutathione (GSH) and F(2)-isoprostanes (F(2)-isoPs). Furthermore, we examined whether mefloquine induces synaptodendritic degeneration of primary rat cortical neurons. GSH was quantified in cortical neurons after 24-h treatment with mefloquine (0, 1, 5, 10 microM) using monochlorobimane. F(2)-isoPs were quantified in cortical neurons after 24-h treatment with mefloquine (0, 1, 5, 10 microM) using a stable isotope dilution method with detection by gas chromatography/mass spectrometry and selective ion monitoring. The concentration dependent decrease in GSH and the concomitant increase of F(2)-isoPs indicates the presence of oxidative stress in primary rat cortical neurons treated with mefloquine. Following a 24-h treatment with mefloquine, primary rat cortical neurons (0, 5, 10 microM) were fixed with 4% paraformaldehyde. Images from eight optical sections covering a distance of 2.88 microm on the z-axis were acquired using a confocal laser scanning unit. Traced images were analyzed with NeuroExplorer, a neurophysiological data analysis package. Mefloquine induces a concentration dependent decrease in the number of spines per neuron and the spine density, suggesting that mefloquine induced oxidative stress may be associated with the synaptodendritic degeneration. Together with previous work, there is strong evidence that a relationship exists between calcium homeostasis disruption, ER stress response, the oxidative stress response, and neurodegeneration. Understanding how oxidative stress alters the morphology of cortical neurons treated with mefloquine will provide further insight into the mechanism(s) related to clinically observed adverse neurological events.

trans-10,cis-12-Conjugated linoleic acid instigates inflammation in human adipocytes compared with preadipocytes.

We showed previously in cultures of primary human adipocytes and preadipocytes that lipopolysaccharide and trans-10,cis-12-conjugated linoleic acid (10,12-CLA) activate the inflammatory signaling that promotes insulin resistance. Because our published data demonstrated that preadipocytes are the primary instigators of inflammatory signaling in lipopolysaccharide-treated cultures, we hypothesized that they played the same role in 10,12-CLA-mediated inflammation. To test this hypothesis, we employed four distinct models. In model 1, a differentiation model, CLA activation of MAPK and induction of interleukin-8 (IL-8), IL-6, IL-1beta, and cyclo-oxygenase-2 (COX-2) were greatest in differentiated compared with undifferentiated cultures. In model 2, a cell separation model, the mRNA levels of these inflammatory proteins were increased by 10,12-CLA compared with bovine serum albumin vehicle in the adipocyte fraction and the preadipocyte fraction. In model 3, a co-culture insert model, inserts containing approximately 50% adipocytes (AD50) or approximately 100% preadipocytes (AD0) were suspended over wells containing AD50 or AD0 cultures. 10,12-CLA-induced IL-8, IL-6, IL-1beta, and COX-2 mRNA levels were highest in AD50 cultures when co-cultured with AD0 inserts. In model 4, a conditioned medium (CM) model, CM collected from CLA-treated AD50 but not AD0 cultures induced IL-8 and IL-6 mRNA levels and activated phosphorylation of MAPK in naive AD0 and AD50 cultures. Consistent with these data, 10,12-CLA-mediated secretions of IL-8 and IL-6 from AD50 cultures were higher than from AD0 cultures. Notably, blocking adipocytokine secretion prevented the inflammatory capacity of CM from 10,12-CLA-treated cultures. These data suggest that CLA instigates the release of inflammatory signals from adipocytes that subsequently activate adjacent preadipocytes.

Methylmercury induces acute oxidative stress, altering Nrf2 protein level in primary microglial cells.

The neurotoxicity of methylmercury (MeHg) is well documented in both humans and animals. MeHg causes acute and chronic damage to multiple organs, most profoundly the central nervous system (CNS). Microglial cells are derived from macrophage cell lineage, making up approximately 12% of cells in the CNS, yet their role in MeHg-induced neurotoxicity is not well defined. The purpose of the present study was to characterize microglial vulnerability to MeHg and their potential adaptive response to acute MeHg exposure. We examined the effects of MeHg on microglial viability, reactive oxygen species (ROS) generation, glutathione (GSH) level, redox homeostasis, and Nrf2 protein expression. Our data showed that MeHg (1-5 microM) treatment caused a rapid (within 1 min) concentration- and time-dependent increase in ROS generation, accompanied by a statistically significant decrease in the ratio of GSH and its oxidized form glutathione disulfide (GSSG) (GSH:GSSG ratio). MeHg increased the cytosolic Nrf2 protein level within 1 min of exposure, followed by its nuclear translocation after 10 min of treatment. Consistent with the nuclear translocation of Nrf2, quantitative real-time PCR revealed a concentration-dependent increase in the messenger RNA level of Ho-1, Nqo1, and xCT 30 min post MeHg exposure, whereas Nrf2 knockdown greatly reduced the upregulation of these genes. Furthermore, we observed increased microglial death upon Nrf2 knockdown by the small hairpin RNA approach. Taken together, our study has demonstrated that microglial cells are exquisitely sensitive to MeHg and respond rapidly to MeHg by upregulating the Nrf2-mediated antioxidant response.