[The Role of Supersulfide in Methylmercury Detoxification].

Methylmercury is a ubiquitous neurotoxic substance present in the environment, and health concerns, especially through the consumption of seafood, remain. Glutathione (GSH)-mediated detoxification and the excretion of methylmercury are known metabolic detoxification pathways. We have also discovered a mechanism by which endogenous super-sulfides convert methylmercury to nontoxic metabolites such as bis-methylmercury sulfide. However, these metabolites are present in very small quantities, and the significance of the detoxification of methylmercury by super-sulfides is not well understood. Methylmercury binds to thiol groups in vivo but can also react with highly reactive selenols (selenocysteine residues). Such covalent bonds (S-mercuration and Se-mercuration) are broken by nucleophilic substitution reactions with other thiol and selenols, however, the contribution of super-sulfides to this substitution reaction is not well understood. Interestingly, a recent study suggested that selenoprotein P, the major selenium transport protein in plasma, binds to methylmercury, however, Se-mercuration was not determined. In this review, we introduce these series of reactions and discuss their involvement with super-sulfides in methylmercury toxicity.

Selenoprotein P expression in glioblastoma as a regulator of ferroptosis sensitivity: preservation of GPX4 via the cycling-selenium storage.

Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.

Sulforaphane decreases serum selenoprotein P levels through enhancement of lysosomal degradation independent of Nrf2.

Selenoprotein P (SeP) is a major selenoprotein in serum predominantly produced in the liver. Excess SeP impairs insulin secretion from the pancreas and insulin sensitivity in skeletal muscle, thus inhibition of SeP could be a therapeutic strategy for type 2 diabetes. In this study, we examine the effect of sulforaphane (SFN), a phytochemical of broccoli sprouts and an Nrf2 activator, on SeP expression in vitro and in vivo. Treatment of HepG2 cells with SFN decreases inter- and intra-cellular SeP levels. SFN enhances lysosomal acidification and expression of V-ATPase, and inhibition of this process cancels the decrease of SeP by SFN. SFN activates Nrf2 in the cells, while Nrf2 siRNA does not affect the decrease of SeP by SFN or lysosomal acidification. These results indicate that SFN decreases SeP by enhancing lysosomal degradation, independent of Nrf2. Injection of SFN to mice results in induction of cathepsin and a decrease of SeP in serum. The findings from this study are expected to contribute to developing SeP inhibitors in the future, thereby contributing to treating and preventing diseases related to increased SeP.

Potential Association between Methylmercury Neurotoxicity and Inflammation.

Methylmercury (MeHg) is the causal substrate of Minamata disease and a major environmental toxicant. MeHg is widely distributed, mainly in the ocean, meaning its bioaccumulation in seafood is a considerable problem for human health. MeHg has been intensively investigated and is known to induce inflammatory responses and neurodegeneration. However, the relationship between MeHg-induced inflammatory responses and neurodegeneration is not understood. In the present review, we first describe recent findings showing an association between inflammatory responses and certain MeHg-unrelated neurological diseases caused by neurodegeneration. In addition, cell-specific MeHg-induced inflammatory responses are summarized for the central nervous system including those of microglia, astrocytes, and neurons. We also describe MeHg-induced inflammatory responses in peripheral cells and tissue, such as macrophages and blood. These findings provide a concept of the relationship between MeHg-induced inflammatory responses and neurodegeneration, as well as direction for future research of MeHg-induced neurotoxicity.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.

Methylmercury directly modifies the 105th cysteine residue in oncostatin M to promote binding to tumor necrosis factor receptor 3 and inhibit cell growth.

We previously found that methylmercury induces expression of oncostatin M (OSM), which is released extracellularly and binds to tumor necrosis factor receptor 3 (TNFR3), possibly enhancing its own toxicity. However, the mechanism by which methylmercury causes OSM to bind to TNFR3 rather than to its known receptors, OSM receptor and LIFR, is unknown. In this study, we aimed to elucidate the effect of methylmercury modification of cysteine residues in OSM on binding to TNFR3. Immunostaining of TNFR3-V5-expressing cells suggested that methylmercury promoted binding of OSM to TNFR3 on the cell membrane. In an in vitro binding assay, OSM directly bound to the extracellular domain of TNFR3, and this binding was promoted by methylmercury. Additionally, the formation of a disulfide bond in the OSM molecule was essential for the binding of both proteins, and LC/MS analysis revealed that methylmercury directly modified the 105th cysteine residue (Cys105) in OSM. Next, mutant OSM, in which Cys105 was replaced by serine or methionine, increased the binding to TNFR3, and a similar effect was observed in immunoprecipitation using cultured cells. Furthermore, cell proliferation was inhibited by treatment with Cys105 mutant OSMs compared with wildtype OSM, and this effect was cancelled by TNFR3 knockdown. In conclusion, we revealed a novel mechanism of methylmercury toxicity, in which methylmercury directly modifies Cys105 in OSM, thereby inhibiting cell proliferation via promoting binding to TNFR3. This indicates a chemical disruption in the interaction between the ligand and the receptor is a part of methylmercury toxicity.

A non-nucleotide agonist that binds covalently to cysteine residues of STING.

Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist.Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide.

Role of selenoprotein P expression in the function of pancreatic ß cells: Prevention of ferroptosis-like cell death and stress-induced nascent granule degradation.

Selenoprotein P (SELENOP) is a major selenium (Se)-containing protein (selenoprotein) in human plasma that is mainly synthesized in the liver. SELENOP transports Se to the cells, while SELENOP synthesized in peripheral tissues is incorporated in a paracrine/autocrine manner to maintain the levels of cellular selenoproteins, called the SELENOP cycle. Pancreatic ß cells, responsible for the synthesis and secretion of insulin, are known to express SELENOP. Here, using MIN6 cells as a mouse model for pancreatic ß cells and Selenop small interfering (si)RNA, we found that Selenop gene knockdown (KD) resulted in decreased cell viability, cellular pro/insulin levels, insulin secretion, and levels of several cellular selenoproteins, including glutathione peroxidase 4 (Gpx4) and selenoprotein K (Selenok). These dysfunctions induced by Selenop siRNA were recovered by the addition of Se. Ferroptosis-like cell death, regulated by Gpx4, was involved in the decrease of cell viability by Selenop KD, while stress-induced nascent granule degradation (SINGD), regulated by Selenok, was responsible for the decrease in proinsulin. SINGD was also observed in the pancreatic ß cells of Selenop knockout mice. These findings indicate a significant role of SELENOP expression for the function of pancreatic ß cells by maintaining the levels of cellular selenoproteins such as GPX4 and SELENOK.

Increased expression of TCF3, transcription factor 3, is a defense response against methylmercury toxicity in mouse neuronal C17.2 cells.

Methylmercury is an environmental pollutant that induces potent neurotoxicity. We previously identified transcription factor 3 (TCF3) as a transcription factor that is activated in the brains of mice treated with methylmercury, and reported that methylmercury sensitivity was increased in cells in which TCF3 expression was suppressed. However, the mechanisms involved in the activation of TCF3 by methylmercury and in the reduction of methylmercury toxicity by TCF3 remained unclear. We found that treatment of mouse neuronal C17.2 cells with methylmercury increased TCF3 protein levels and promoted the binding of TCF3 to DNA consensus sequences. In cells treated with actinomycin D, a transcription inhibitor, an increase in TCF3 protein levels was also observed under methylmercury exposure. However, in the presence of cycloheximide, a translation inhibitor, methylmercury delayed the degradation of TCF3 protein. In addition, treatment with MG132, a proteasome inhibitor, increased TCF3 protein levels, and there was not significant increase in TCF3 protein levels by methylmercury under these conditions. These results suggest that methylmercury may activate TCF3 by increasing its levels through inhibition of TCF3 degradation by the proteasome. It has been previously reported that the induction of apoptosis in neurons is involved in methylmercury-induced neuronal damage in the brain. Although apoptosis was induced in C17.2 cells treated with methylmercury, this induction was largely suppressed by overexpression of TCF3. These results indicate that TCF3, which is increased in the brain upon exposure to methylmercury, may be a novel defense factor against methylmercury-induced neurotoxicity.

Identification of a novel endogenous long non-coding RNA that inhibits selenoprotein P translation.

Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3' untranslated region (3'UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3'UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA.

Methylmercury induces neuronal cell death by inducing TNF-α expression through the ASK1/p38 signaling pathway in microglia.

We recently found that tumor necrosis factor-α (TNF-α) may be involved in neuronal cell death induced by methylmercury in the mouse brain. Here, we examined the cells involved in the induction of TNF-α expression by methylmercury in the mouse brain by in situ hybridization. TNF-α-expressing cells were found throughout the brain and were identified as microglia by immunostaining for ionized calcium binding adaptor molecule 1 (Iba1). Methylmercury induced TNF-α expression in mouse primary microglia and mouse microglial cell line BV2. Knockdown of apoptosis signal-regulating kinase 1 (ASK1), an inflammatory cytokine up-regulator that is responsible for reactive oxygen species (ROS), decreased methylmercury-induced TNF-α expression through decreased phosphorylation of p38 MAP kinase in BV2 cells. Suppression of methylmercury-induced reactive oxygen species (ROS) by antioxidant treatment largely abolished the induction of TNF-α expression and phosphorylation of p38 by methylmercury in BV2 cells. Finally, in mouse brain slices, the TNF-α antagonist (WP9QY) inhibited neuronal cell death induced by methylmercury, as did the p38 inhibitor SB203580 and liposomal clodronate (a microglia-depleting agent). These results indicate that methylmercury induces mitochondrial ROS that are involved in activation of the ASK1/p38 pathway in microglia and that this is associated with induction of TNF-α expression and neuronal cell death.

Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveal the effects of multiple oxidation on cellular oxidative stress response.

Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H2O2). Possibly due to obstruction of the dimer-dimer interface, H2O2-induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (-Sn-, n ≧ 3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2C358,424A) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs compared with cells expressing wild-type PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress.

Gefitinib initiates sterile inflammation by promoting IL-1ß and HMGB1 release via two distinct mechanisms.

Anticancer drug gefitinib causes inflammation-based side effects, such as interstitial pneumonitis. However, its mechanisms remain unknown. Here, we provide evidence that gefitinib elicits pro-inflammatory responses by promoting mature-interleukin-1ß (IL-1ß) and high-mobility group box 1 (HMGB1) release. Mitochondrial reactive oxygen species (mtROS) driven by gefitinib stimulated the formation of the NLRP3 (NACHT, LRR and PYD-containing protein 3) inflammasome, leading to mature-IL-1ß release. Notably, gefitinib also stimulated HMGB1 release, which is, however, not mediated by the NLRP3 inflammasome. On the other hand, gefitinib-driven mtROS promoted the accumulation of γH2AX, a hallmark of DNA damage, leading to the activation of poly (ADP-ribose) polymerase-1 (PARP-1) and subsequent active release of HMGB1. Together our results reveal the potential ability of gefitinib to initiate sterile inflammation via two distinct mechanisms, and identified IL-1ß and HMGB1 as key determinants of gefitinib-induced inflammation that may provide insights into gefitinib-induced interstitial pneumonitis.

Hydrogen Peroxide Causes Cell Death via Increased Transcription of HOXB13 in Human Lung Epithelial A549 Cells.

Although homeobox protein B13 (HOXB13) is an oncogenic transcription factor, its role in stress response has rarely been examined. We previously reported that knockdown of HOXB13 reduces the cytotoxicity caused by various oxidative stress inducers. Here, we studied the role of HOXB13 in cytotoxicity caused by hydrogen peroxide in human lung epithelial A549 cells. The knockdown of HOXB13 reduced hydrogen peroxide-induced cytotoxicity; however, this phenomenon was largely absent in the presence of antioxidants (Trolox or N-acetyl cysteine (NAC)). This suggests that HOXB13 may be involved in the cytotoxicity caused by hydrogen peroxide via the production of reactive oxygen species (ROS). Hydrogen peroxide also increased both the mRNA and protein levels of HOXB13. However, these increases were rarely observed in the presence of a transcriptional inhibitor, which suggests that hydrogen peroxide increases protein levels via increased transcription of HOXB13. Furthermore, cell death occurred in A549 cells that highly expressed HOXB13. However, this cell death was mostly inhibited by treatment with antioxidants. Taken together, our findings indicate that HOXB13 may be a novel factor involved in the induction of oxidative stress, which causes cell death via intracellular ROS production.

Elaidic Acid Potentiates Extracellular ATP-Induced Apoptosis via the P2X7-ROS-ASK1-p38 Axis in Microglial Cell Lines.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X7), or Ca2+/calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

Increased putrescine levels due to ODC1 overexpression prevents mitochondrial dysfunction-related apoptosis induced by methylmercury.

AIMS: We had previously reported that addition of putrescine to the culture medium was reported to reduce methylmercury toxicity in C17.2 neural stem cells. Here, we have examined the inhibition of methylmercury-induced cytotoxicity by putrescine using ODC1-overexpressing C17.2 cells. MATERIALS AND METHODS: We established stable ODC1-overexpressing C17.2 cells and evaluated methylmercury-induced apoptosis by examining the TUNEL assay and cleaved caspase-3 levels. Mitochondria-mediated apoptosis was also evaluated by reduction of mitochondrial membrane potential and recruitment of Bax and Bak to the mitochondria. KEY FINDINGS: ODC is encoded by ODC1 gene, and putrescine levels in ODC1-overexpressing cells were significantly higher than in control cells. Overexpression of ODC1 and addition of putrescine to the culture medium suppressed DNA fragmentation and caspase-3 activation, which are observed when apoptosis is induced by methylmercury. Moreover, mitochondrial dysfunction and reactive oxygen species (ROS) generation, caused by methylmercury, were also inhibited by the overexpression of ODC1 and putrescine; pretreatment with ODC inhibitor, however, promoted both ROS generation and apoptosis by methylmercury. Finally, we found that Bax and Bak, the apoptosis-promoting factors, to be increased in mitochondria, following methylmercury treatment, and the same was inhibited by overexpression of ODC1. These results suggest that overexpression of ODC1 may prevent mitochondria-mediated apoptosis by methylmercury via increase of putrescine levels. SIGNIFICANCE: Our findings provide important clues to clarify mechanisms involved in the defense against methylmercury toxicity and suggest novel biological functions of putrescine.

The Nuclear Protein HOXB13 Enhances Methylmercury Toxicity by Inducing Oncostatin M and Promoting Its Binding to TNFR3 in Cultured Cells.

Homeobox protein B13 (HOXB13), a transcription factor, is related to methylmercury toxicity; however, the downstream factors involved in enhancing methylmercury toxicity remain unknown. We performed microarray analysis to search for downstream factors whose expression is induced by methylmercury via HOXB13 in human embryonic kidney cells (HEK293), which are useful model cells for analyzing molecular mechanisms. Methylmercury induced the expression of oncostatin M (OSM), a cytokine of the interleukin-6 family, and this was markedly suppressed by HOXB13 knockdown. OSM knockdown also conferred resistance to methylmercury in HEK293 cells, and no added methylmercury resistance was observed when both HOXB13 and OSM were knocked down. Binding of HOXB13 to the OSM gene promoter was increased by methylmercury, indicating the involvement of HOXB13 in the enhancement of its toxicity. Because addition of recombinant OSM to the medium enhanced methylmercury toxicity in OSM-knockdown cells, extracellularly released OSM was believed to enhance methylmercury toxicity via membrane receptors. We discovered tumor necrosis factor receptor (TNF) receptor 3 (TNFR3) to be a potential candidate involved in the enhancement of methylmercury toxicity by OSM. This toxicity mechanism was also confirmed in mouse neuronal stem cells. We report, for the first time, that HOXB13 is involved in enhancement of methylmercury toxicity via OSM-expression induction and that the synthesized OSM causes cell death by binding to TNFR3 extracellularly.

Induction of chemokine CCL3 by NF-κB reduces methylmercury toxicity in C17.2 mouse neural stem cells.

Methylmercury is an environmental pollutant that shows selective toxicity to the central nervous system. We previously reported that brain-specific expression of chemokine CCL3 increases in mice administered methylmercury. However, the relationship between CCL3 and methylmercury toxicity has not been elucidated. Here, we confirmed that induction of CCL3 expression occurs before pathological change by methylmercury treatment was observed in the mouse brain. This induction was also observed in C17.2 mouse neural stem cells before methylmercury-induced cytotoxicity. In addition, cells in which CCL3 was knocked-down showed higher methylmercury sensitivity than did control cells. Moreover, activation of transcription factor NF-κB was observed following methylmercury treatment, and methylmercury-mediated induction of CCL3 expression was partially suppressed by knockdown of p65, an NF-κB subunit. Our results suggest that NF-κB plays a role in the induction of methylmercury-mediated CCL3 expression and that this action may be a cellular response to methylmercury toxicity.

Evaluation of M1-microglial activation by neurotoxic metals using optimized organotypic cerebral slice cultures.

M1-microglia (neurotoxic microglia) regulate neuronal development and cell death and are involved in many pathologies in the brain. Although organotypic brain slice cultures are widely used to study the crosstalk between neurons and microglia, little is known about the properties of microglia in the mouse cerebral cortex slices. Here, we aimed to optimize the mouse cerebral slice cultures that reflect microglial functions and evaluate the effects of neurotoxic metals on M1-microglial activation. Most microglia in the cerebral slices prepared from postnatal day (P) 7 mice were similar to mature microglia in adult mice brains, but those in the slices prepared from P2 mice were immature, which is a conventional preparation condition. The degree of expression of M1-microglial markers (CD16 and CD32) and inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) by lipopolysaccharide, a representative microglia activator, in the cerebral slices of P7 mice were higher than that in the slices of P2 mice. These results indicate that M1-microglial activation can be evaluated more accurately in the cerebral slices of P7 mice than in those of P2 mice. Therefore, we next examined the effects of various neurotoxic metals on M1-microglial activation using the cerebral slices of P7 mice and found that methylmercury stimulated the activation to M1-microglia, but arsenite, lead, and tributyltin did not induce such activation. Altogether, the optimized mouse cerebral slice cultures used in this study can be a helpful tool to study the influence of various chemicals on the central nervous system in the presence of functionally mature microglia.