Manganese Is Essential for Neuronal Health.

The understanding of manganese (Mn) biology, in particular its cellular regulation and role in neurological disease, is an area of expanding interest. Mn is an essential micronutrient that is required for the activity of a diverse set of enzymatic proteins (e.g., arginase and glutamine synthase). Although necessary for life, Mn is toxic in excess. Thus, maintaining appropriate levels of intracellular Mn is critical. Unlike other essential metals, cell-level homeostatic mechanisms of Mn have not been identified. In this review, we discuss common forms of Mn exposure, absorption, and transport via regulated uptake/exchange at the gut and blood-brain barrier and via biliary excretion. We present the current understanding of cellular uptake and efflux as well as subcellular storage and transport of Mn. In addition, we highlight the Mn-dependent and Mn-responsive pathways implicated in the growing evidence of its role in Parkinson's disease and Huntington's disease. We conclude with suggestions for future focuses of Mn health-related research.

Dopaminergic neurotoxicity of S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, and S-methyl-N,N-diethylthiocarbamate (MeDETC) in Caenorhabditis elegans.

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S-ethyl N,N-dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S-methyl-N,N-diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic-dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.

Involvement of AAT transporters in methylmercury toxicity in Caenorhabditis elegans.

Methylmercury (MeHg) is a potent neurotoxin that enters mammalian cells as a conjugate with L-cysteine through L-type large neutral amino acid transporter, LAT1, by a molecular mimicry mechanism by structurally resembling L-methionine. Caenorhabditis elegans (C. elegans) has been increasingly used to study the neurotoxic effects of MeHg, but little is known about uptake and transport of MeHg in the worm. This study examined whether MeHg uptake through LAT1 is evolutionarily conserved in nematodes. MeHg toxicity in C. elegans was blocked by pre-treatment of worms with l-methionine, suggesting a role for amino acid transporters in MeHg transport. Knockdown of aat-1, aat-2, and aat-3, worm homologues to LAT1, increased the survival of C. elegans following MeHg treatment and significantly attenuated MeHg content following exposure. These results indicate that MeHg is transported in the worm by a conserved mechanism dependent on functioning amino acid transporters.

Electrophilic adduction of ubiquitin activating enzyme E1 by N,N-diethyldithiocarbamate inhibits ubiquitin activation and is accompanied by striatal injury in the rat.

Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN(2) chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo, and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response, and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, and enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally, this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.

Methylmercury-Induced Metabolic Alterations in Caenorhabditis elegans Are Diet-Dependent.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. Chronic exposure to MeHg in human populations shows an association with diabetes mellitus and metabolic syndrome (MS). As the incidences of both obesity and MS are on the rise globally, it is important to understand the potential role of MeHg in the development of the disease. There is a dearth of information on dietary interactions between MeHg and lipids, which play an important role in developing MS. We have previously shown that MeHg increases food seeking behaviors, lipid levels, fat storage, and pro-adipogenic gene expression in C. elegans fed the standard OP50 Escherichia coli diet. However, we hypothesized that these metabolic changes could be prevented if the worms were fed a bacterial diet lower in lipid content. We tested whether C. elegans developed metabolic alterations in response to MeHg if they were fed two alternative E. coli strains (HT115 and HB101) that are known absorb significantly less lipids from their media. Additionally, to explore the effect of a high-lipid and high-cholesterol diet on MeHg-induced metabolic dysfunction, we supplemented the OP50 strain with twice the standard concentration of cholesterol in the nematode growth media. Wild-type worms fed either the HB101 or HT115 diet were more resistant to MeHg than the worms fed the OP50 diet, showing a significant right-hand shift in the dose-response survival curve. Worms fed the OP50 diet supplemented with cholesterol were more sensitive to MeHg, showing a significant left-hand shift in the dose-response survival curve. Changes in sensitivity to MeHg by differential diet were not due to altered MeHg intake in the worms as measured by inductively coupled mass spectrometry. Worms fed the low-fat diets showed protection from MeHg-induced metabolic changes, including decreased food consumption, lower triglyceride content, and lower fat storage than the worms fed either of the higher-fat diets. Oxidative stress is a common characteristic of both MeHg exposure and high-fat diets. Worms fed either OP50 or OP50 supplemented with cholesterol and treated with MeHg had significantly higher levels of reactive oxygen species, carbonylated proteins, and loss of glutathione than the worms fed the HT115 or HB101 low-lipid diets. Taken together, our data suggest a synergistic effect of MeHg and dietary lipid levels on MeHg toxicity and fat metabolism in C. elegans, which may affect the ability of MeHg to cause metabolic dysfunction.

Methylmercury Induces Metabolic Alterations in Caenorhabditis elegans: Role for C/EBP Transcription Factor.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. We have previously shown that MeHg causes metabolic alterations in Caenorhabditis elegans, leading to decreased nicotinamide adenine dinucleotide cofactor, mitochondrial dysfunction, and oxidative stress. We were, therefore, interested in whether MeHg also affects nutrient metabolism, particularly lipid homeostasis, which may contribute to the development of metabolic conditions such as obesity or metabolic syndrome (MS). RNA from wild-type worms exposed to MeHg was collected immediately after treatment and used for gene expression analysis by DNA microarray. MeHg differentially regulated 215 genes, 17 genes involved in lipid homeostasis, and 12 genes involved in carbohydrate homeostasis. Of particular interest was cebp-1, the worm ortholog to human C/EBP, a pro-adipogenic transcription factor implicated in MS. MeHg increased the expression of cebp-1 as well as pro-adipogenic transcription factors sbp-1 and nhr-49, triglyceride synthesis enzyme acl-6, and lipid transport proteins vit-2 and vit-6. Concurrent with the altered gene expression, MeHg increased triglyceride levels, lipid storage, and feeding behaviors. Worms expressing mutant cebp-1 were protected from MeHg-induced alterations in lipid content, feeding behaviors, and gene expression, highlighting the importance of this transcription factor in the worm's response to MeHg. Taken together, our data demonstrate that MeHg induces biochemical, metabolic, and behavioral changes in C. elegans that can lead to metabolic dysfunction.

Variation in the biological half-life of methylmercury in humans: Methods, measurements and meaning.

BACKGROUND: Understanding methylmercury (MeHg) toxicity requires a complete understanding of its fundamental toxicokinetic and toxicodynamic characteristics in the human body. The biological half-life (t1/2) of MeHg is a kinetic property that directly influences the body burden of Hg that results from repeated exposures such as can occur with fish and seafood consumption. The t1/2 of MeHg in humans is approximately 50 days, equivalent to an elimination rate (kel) of 0.014 day-1. However, numerous studies report a wide range of half-life values (t1/2 < 30 to >120 days), demonstrating that significant variation in the biological process of MeHg elimination exists. This variation is a source of considerable uncertainty in deriving a meaningful reference dose for MeHg applicable to all individuals in a population. SCOPE OF REVIEW: First, we summarize fundamentals of MeHg toxicokinetics, emphasizing the central role that biological half-life plays in MeHg dosimetry. We next present important considerations for how kinetic analyses are performed. We provide an example of how MeHg half-life variation directly influences the body burden and, in certain contexts, can result in MeHg levels exceeding the US EPA Reference Dose. We then survey existing studies that report MeHg half-life determinations in people. MAJOR CONCLUSIONS: Recent advances in methods of determining MeHg kinetics in people have made individualized assessment of MeHg elimination rates more accurate and readily obtainable. GENERAL SIGNIFICANCE: Characterization of MeHg half-life, particularly in vulnerable individuals, such as pregnant women and children, will diminish the remaining toxicokinetic uncertainty surrounding MeHg exposures and will better inform the risk assessment process.

Editor's Highlight: Variation in Methylmercury Metabolism and Elimination Status in Humans Following Fish Consumption.

Evaluating the potential for methylmercury (MeHg) toxicity relies on accurately predicting the mercury (Hg) body burden that results from eating fish. Hg body burden is directly determined by the slow elimination kinetics of MeHg in the human body (kel = 0.014 days-1 or t1/2 =50 days). Existing studies on MeHg half-life in humans demonstrate a wide range values (t1/2 = 30 to >150 days) and has lead to uncertainty in the derivation of a regulatory standard for acceptable daily oral intake. The causes of variation in MeHg toxicokinetics in humans remain little explored. Here we characterize variation in human MeHg metabolism and elimination rate (kel) in 37 adult volunteers who consumed 3 fish meals. We determined MeHg elimination rates via longitudinal Hg analysis in single hairs using laser ablation inductively coupled plasma mass spectrometry. We also measured MeHg metabolism (biotransformation) via speciation of fecal Hg. We find an average kel = 0.0157 days-1 (t1/2 = 44 days) amongst a more than 2-fold variation in kel across the cohort (0.0248-0.0112 days-1; t1/2 = 28-62 days). Although MeHg biotransformation varied widely between individuals, it showed a positive association with elimination rates across the cohort. A more than 2-fold change in kel over a period of 2 years was seen in some individuals. In 2 individuals, who received antibiotic for unrelated health issues, elimination rate was seen to slow significantly. Associations of kel with age, body mass index, gender, and fish eating habits were not observed. We establish that a measure of methylmercury metabolism and eliminaiton status (MerMES) can reduce uncertainty in determining an individual's MeHg toxicokinetics subsequent to eating fish.

NAD+ Supplementation Attenuates Methylmercury Dopaminergic and Mitochondrial Toxicity in Caenorhabditis Elegans.

Methylmercury (MeHg) is a neurotoxic contaminant of our fish supply that has been linked to dopaminergic (DAergic) dysfunction that characterizes Parkinson's disease. We have previously shown that MeHg causes both morphological and behavioral changes in the Caenorhabditis elegans DAergic neurons that are associated with oxidative stress. We were therefore interested in whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD(+)) may be affected by MeHg and whether supplementation of NAD( + )may prevent MeHg-induced toxicities. Worms treated with MeHg showed depletion in cellular NAD( + )levels, which was prevented by NAD( + )supplementation prior to MeHg treatment. NAD( + )supplementation also prevented DAergic neurodegeneration and deficits in DAergic-dependent behavior upon MeHg exposure. In a mutant worm line that cannot synthesize NAD( + )from nicotinamide, MeHg lethality and DAergic behavioral deficits were more sensitive to MeHg than wildtype worms, demonstrating the importance of NAD( + )in MeHg toxicity. In wildtype worms, NAD( + )supplementation provided protection from MeHg-induced oxidative stress and mitochondrial dysfunction. These data show the importance of NAD( + )levels in the response to MeHg exposure. NAD( + )supplementation may be beneficial for MeHg-induced toxicities and preventing cellular damage involved in Parkinson's disease.

Scavengers of reactive γ-ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7.

Isoketals (IsoKs) are highly reactive γ-ketoaldehyde products of lipid peroxidation that covalently adduct lysine side chains in proteins, impairing their function. Using C. elegans as a model organism, we sought to test the hypothesis that IsoKs contribute to molecular aging through adduction and inactivation of specific protein targets, and that this process can be abrogated using salicylamine (SA), a selective IsoK scavenger. Treatment with SA extends adult nematode longevity by nearly 56% and prevents multiple deleterious age-related biochemical and functional changes. Testing of a variety of molecular targets for SA's action revealed the sirtuin SIR-2.1 as the leading candidate. When SA was administered to a SIR-2.1 knockout strain, the effects on lifespan and healthspan extension were abolished. The SIR-2.1-dependent effects of SA were not mediated by large changes in gene expression programs or by significant changes in mitochondrial function. However, expression array analysis did show SA-dependent regulation of the transcription factor ets-7 and associated genes. In ets-7 knockout worms, SA's longevity effects were abolished, similar to sir-2.1 knockouts. However, SA dose-dependently increases ets-7 mRNA levels in non-functional SIR-2.1 mutant, suggesting that both are necessary for SA's complete lifespan and healthspan extension.

Quantification of Glutathione in Caenorhabditis elegans.

Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5'-thio-2-nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains.

Mitochondrial Redox Dysfunction and Environmental Exposures.

SIGNIFICANCE: Mitochondria are structurally and biochemically diverse, even within a single type of cell. Protein complexes localized to the inner mitochondrial membrane synthesize ATP by coupling electron transport and oxidative phosphorylation. The organelles produce reactive oxygen species (ROS) from mitochondrial oxygen and ROS can, in turn, alter the function and expression of proteins used for aerobic respiration by post-translational and transcriptional regulation. RECENT ADVANCES: New interest is emerging not only into the roles of mitochondria in disease development and progression but also as a target for environmental toxicants. CRITICAL ISSUES: Dysregulation of respiration has been linked to cell death and is a major contributor to acute neuronal trauma, peripheral diseases, as well as chronic neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. FUTURE DIRECTIONS: Here, we discuss the mechanisms underlying the sensitivity of the mitochondrial respiratory complexes to redox modulation, as well as examine the effects of environmental contaminants that have well-characterized mitochondrial toxicity. The contaminants discussed in this review are some of the most prevalent and potent environmental contaminants that have been linked to neurological dysfunction, altered cellular respiration, and oxidation.

Differential inflammatory response to acrylonitrile in rat primary astrocytes and microglia.

Acrylonitrile (ACN) is extensively used in the production of plastics, resins, nitriles and other commercial products. Chronic low dose exposures to ACN cause glial cell tumors in rats, primarily microglial in origin. Recently it has been determined that astrocytes and microglia respond to ACN-induced oxidative stress differently, which may influence cell-specific activation of inflammatory and carcinogenic pathways. This study was conducted to compare the inflammatory responses of astrocytes and microglia following ACN treatment in vitro to further characterize differential sensitivities and adaptive responses in these cell types. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p53 levels were measured along with levels of 12 different cytokines and chemokines in primary rat microglia and astrocytes. Additionally levels of cytochrome P450 2E1 (CYP2E1) were measured to evaluate the cells' ability to metabolize ACN. Results indicate that while both cells upregulate p53 and NF-κB, the cytokines and chemokines produced differ between the cell types. Astrocytes, but not microglia, upregulated CYP2E1 in response to ACN, which may be due to the astrocytes accumulating more ACN than the microglia. Altogether our data implicate the inflammatory response as an important event in ACN-induced neurotoxicity.

Activation of MAPK and FoxO by manganese (Mn) in rat neonatal primary astrocyte cultures.

Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced 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.