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Hijacking microglial glutathione by inorganic arsenic impels bystander death of immature neurons through extracellular cystine/glutamate imbalance.

ABSTRACT: Arsenic-induced altered microglial activity leads to neuronal death, but the causative mechanism remains unclear. The present study showed, arsenic-exposed (10??M) microglial (N9) culture supernatant induced bystander death of neuro-2a (N2a), which was further validated with primary microglia and immature neuronal cultures. Results indicated that arsenic-induced GSH synthesis by N9 unfavorably modified the extracellular milieu for N2a by lowering cystine and increasing glutamate concentration. Similar result was observed in N9-N2a co-culture. Co-exposure of arsenic and 250??M glutamate, less than the level (265??M) detected in arsenic-exposed N9 culture supernatant, compromised N2a viability which was rescued by cystine supplementation. Therefore, microglia executes bystander N2a death by competitive inhibition of system Xc(-) (xCT) through extracellular cystine/glutamate imbalance. We confirmed the role of xCT in mediating bystander N2a death by siRNA inhibition studies. Ex-vivo primary microglia culture supernatant from gestationally exposed mice measured to contain lower cystine and higher glutamate compared to control and N-acetyl cysteine co-treated group. Immunofluorescence staining of brain cryosections from treated group showed more dead immature neurons with no such effect on microglia. Collectively, we showed, in presence of arsenic microglia alters cystine/glutamate balance through xCT in extracellular milieu leading to bystander death of immature neurons.

SUBMITTER: Singh V

PROVIDER: S-EPMC4967897 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Hijacking microglial glutathione by inorganic arsenic impels bystander death of immature neurons through extracellular cystine/glutamate imbalance.

Singh Vikas V Gera Ruchi R Kushwaha Rajesh R Sharma Anuj Kumar AK Patnaik Satyakam S Ghosh Debabrata D

Scientific reports 20160801

Arsenic-induced altered microglial activity leads to neuronal death, but the causative mechanism remains unclear. The present study showed, arsenic-exposed (10 μM) microglial (N9) culture supernatant induced bystander death of neuro-2a (N2a), which was further validated with primary microglia and immature neuronal cultures. Results indicated that arsenic-induced GSH synthesis by N9 unfavorably modified the extracellular milieu for N2a by lowering cystine and increasing glutamate concentration. S ...[more]

PMID: 27477106

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Project description:Arsenic occurs naturally in the environment, and exists predominantly as inorganic arsenite (As (III) and arsenate As (V)). Arsenic contamination of drinking water has long been recognized as a major global health concern. Arsenic exposure causes changes in skin color and lesions, and more severe health conditions such as black foot disease as well as various cancers originating in the lungs, skin, and bladder. In order to efficiently metabolize and excrete arsenic, it is methylated to monomethylarsonic and dimethylarsinic acid. One single enzyme, arsenic methyltransferase (AS3MT) is responsible for generating both metabolites. AS3MT has been purified from several mammalian and nonmammalian species, and its mRNA sequences were determined from amino acid sequences. With the advent of genome technology, mRNA sequences of AS3MT have been predicted from many species throughout the animal kingdom. Horizontal gene transfer had been postulated for this gene through phylogenetic studies, which suggests the importance of this gene in appropriately handling arsenic exposures in various organisms. An altered ability to methylate arsenic is dependent on specific single nucleotide polymorphisms (SNPs) in AS3MT. Reduced AS3MT activity resulting in poor metabolism of iAs has been shown to reduce expression of the tumor suppressor gene, p16, which is a potential pathway in arsenic carcinogenesis. Arsenic is also known to induce oxidative stress in cells. However, the presence of antioxidant response elements (AREs) in the promoter sequences of AS3MT in several species does not correlate with the ability to methylate arsenic. ARE elements are known to bind NRF2 and induce antioxidant enzymes to combat oxidative stress. NRF2 may be partly responsible for the biotransformation of iAs and the generation of methylated arsenic species via AS3MT. In this article, arsenic metabolism, excretion, and toxicity, a discussion of the AS3MT gene and its evolutionary history, and DNA methylation resulting from arsenic exposure have been reviewed.

Project description:The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.

Project description:Following an official request to EFSA from the European Commission, EFSA assessed the chronic dietary exposure to inorganic arsenic (iAs) in the European population. A total of 13,608 analytical results on iAs were considered in the current assessment (7,623 corresponding to drinking water and 5,985 to different types of food). Samples were collected across Europe between 2013 and 2018. The highest mean dietary exposure estimates at the lower bound (LB) were in toddlers (0.30 ?g/kg body weight (bw) per day), and in both infants and toddlers (0.61 ?g/kg bw per day) at the upper bound (UB). At the 95th percentile, the highest exposure estimates (LB-UB) were 0.58 and 1.20 ?g/kg bw per day in toddlers and infants, respectively. In general, UB estimates were two to three times higher than LB estimates. The mean dietary exposure estimates (LB) were overall below the range of benchmark dose lower confidence limit (BMDL 01) values of 0.3-8 ?g/kg bw per day established by the EFSA Panel on Contaminants in the Food Chain in 2009. However, for the 95th percentile dietary exposure (LB), the maximum estimates for infants, toddlers and other children were within this range of BMDL 01 values. Across the different age classes, the main contributors to the dietary exposure to iAs (LB) were 'Rice', 'Rice-based products', 'Grains and grain-based products (no rice)' and 'Drinking water'. Different ad hoc exposure scenarios (e.g. consumption of rice-based formulae) showed dietary exposure estimates in average and for high consumers close to or within the range of BMDL 01 values. The main uncertainties associated with the dietary exposure estimations refer to the impact of using the substitution method to treat the left-censored data (LB-UB differences), to the lack of information (consumption and occurrence) on some iAs-containing ingredients in specific food groups, and to the effect of food preparation on the iAs levels. Recommendations were addressed to improve future dietary exposure assessments to iAs.

Dataset Information

Hijacking microglial glutathione by inorganic arsenic impels bystander death of immature neurons through extracellular cystine/glutamate imbalance.

Publications

Hijacking microglial glutathione by inorganic arsenic impels bystander death of immature neurons through extracellular cystine/glutamate imbalance.

Similar Datasets

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