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.

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.

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.

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.

Methylmercury toxic mechanism related to protein degradation and chemokine transcription.

Methylmercury is an environmental pollutant that causes neurotoxicity. Recent studies have reported that the ubiquitin-proteasome system is involved in defense against methylmercury toxicity through the degradation of proteins synthesizing the pyruvate. Mitochondrial accumulation of pyruvate can enhance methylmercury toxicity. In addition, methylmercury exposure induces several immune-related chemokines, specifically in the brain, and may cause neurotoxicity. This summary highlights several molecular mechanisms of methylmercury-induced neurotoxicity.

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.

Methylmercury induces the expression of chemokine CCL4 via SRF activation in C17.2 mouse neural stem cells.

Methylmercury is an environmental pollutant that causes specific and serious damage to the central nervous system. We have previously shown that C-C motif chemokine ligand 4 (CCL4) protects cultured neural cells from methylmercury toxicity and expression of CCL4 is specifically induced in mouse brain by methylmercury. In this study, we examined the transcriptional regulatory mechanism that induces CCL4 expression by methylmercury using C17.2 mouse neural stem cells. The promoter region of the CCL4 gene was analyzed by a reporter assay, revealing that the region up to 50 bp upstream from the transcription start site was necessary for inducing expression of CCL4 by methylmercury. Nine transcription factors that might bind to this upstream region and be involved in the induction of CCL4 expression by methylmercury were selected, and the induction of CCL4 expression by methylmercury was suppressed by the knockdown of serum response factor (SRF). In addition, the nuclear level of SRF was elevated by methylmercury, and an increase in the amount bound to the CCL4 gene promoter was also observed. Furthermore, we examined the upstream signaling pathway involved in the induction of CCL4 expression by SRF, and confirmed that activation of p38 and ERK, which are part of the MAPK pathway, are involved. These results suggest that methylmercury induces the expression of CCL4 by activating SRF via the p38 and ERK signaling pathway. Our findings are important for elucidating the mechanism involved in the brain-specific induction of CCL4 expression by methylmercury.

Effect of Metallothionein-III on Mercury-Induced Chemokine Gene Expression.

Mercury compounds are known to cause central nervous system disorders; however the detailed molecular mechanisms of their actions remain unclear. Methylmercury increases the expression of several chemokine genes, specifically in the brain, while metallothionein-III (MT-III) has a protective role against various brain diseases. In this study, we investigated the involvement of MT-III in chemokine gene expression changes in response to methylmercury and mercury vapor in the cerebrum and cerebellum of wild-type mice and MT-III null mice. No difference in mercury concentration was observed between the wild-type mice and MT-III null mice in any brain tissue examined. The expression of Ccl3 in the cerebrum and of Cxcl10 in the cerebellum was increased by methylmercury in the MT-III null but not the wild-type mice. The expression of Ccl7 in the cerebellum was increased by mercury vapor in the MT-III null mice but not the wild-type mice. However, the expression of Ccl12 and Cxcl12 was increased in the cerebrum by methylmercury only in the wild-type mice and the expression of Ccl3 in the cerebellum was increased by mercury vapor only in the wild-type mice. These results indicate that MT-III does not affect mercury accumulation in the brain, but that it affects the expression of some chemokine genes in response to mercury compounds.

Activation of ornithine decarboxylase protects against methylmercury toxicity by increasing putrescine.

We previously reported significantly increased level of putrescine, a polyamine, in the brains of mice administered methylmercury. Moreover, addition of putrescine to culture medium reduced methylmercury toxicity in C17.2 mouse neural stem cells. In this study, the role of ornithine decarboxylase (ODC), an enzyme involved in putrescine synthesis, in response to methylmercury toxicity was investigated. Methylmercury increased ODC activity in mouse cerebrum and cerebellum, but this increase was hardly observed in the kidney and liver, where methylmercury accumulated at a high concentration. In the cerebrum and cerebellum, increased putrescine was observed with methylmercury administration. Methylmercury increased ODC activity in C17.2 cells, but this was almost completely abolished in the presence of an ODC inhibitor. Methylmercury also increased the level of ODC protein in mouse brain and C17.2 cells. In addition, C17.2 cells pretreated with ODC inhibitor showed higher methylmercury sensitivity than control cells. These results suggest that the increased ODC activity by methylmercury is involved in the increase in putrescine level, and ODC plays an important role in the reduction of methylmercury toxicity. This is the first study to provide evidence that increased ODC activity may be a protective response against methylmercury-induced neurotoxicity.

Chemokine CCL4 Induced in Mouse Brain Has a Protective Role against Methylmercury Toxicity.

Methylmercury (MeHg) is selectively toxic to the central nervous system, but mechanisms related to its toxicity are poorly understood. In the present study, we identified the chemokine, C-C motif Chemokine Ligand 4 (CCL4), to be selectively upregulated in the brain of MeHg-administered mice. We then investigated the relationship between CCL4 expression and MeHg toxicity using in vivo and in vitro approaches. We confirmed that in C17.2 cells (a mouse neural stem cell line) and the mouse brain, induction of CCL4 expression occurs prior to cytotoxicity caused by MeHg. We also show that the addition of recombinant CCL4 to the culture medium of mouse xprimary neurons attenuated MeHg toxicity, while knockdown of CCL4 in C17.2 cells resulted in higher MeHg sensitivity compared with control cells. These results suggest that CCL4 is a protective factor against MeHg toxicity and that induction of CCL4 expression is not a result of cytotoxicity by MeHg but is a protective response against MeHg exposure.

Akr1 attenuates methylmercury toxicity through the palmitoylation of Meh1 as a subunit of the yeast EGO complex.

BACKGROUND: We previously reported that palmitoyltransferase activity of Akr1 is required for alleviation of methylmercury toxicity in yeast. In this study, we identified a factor that alleviates methylmercury toxicity among the substrate proteins palmitoylated by Akr1, and investigated the role of this factor in methylmercury toxicity. METHODS: Gene disruption and site-directed mutagenesis were used to examine the relationship of methylmercury toxicity and vacuole function. Palmitoylation was investigated using the acyl-biotinyl exchange method. Vacuoles were stained with the fluorescent probe FM4-64. RESULTS: We found that Meh1 (alias Ego1), a substrate protein of Akr1, participates in the alleviation of methylmercury toxicity. Moreover, almost no palmitoylation of Meh1 when Akr1 was knocked out, and mutant Meh1, which is not palmitoylated, did not show alleviation of methylmercury toxicity. The palmitoylated Meh1 was involved in the alleviation of methylmercury toxicity as a constituent of EGO complex which suppresses autophagy. Methylmercury caused vacuole deformation, and this was greater in the yeasts knocking out the EGO complex subunits. 3-Methyladenine, an autophagy inhibitor, suppresses vacuole deformation and cytotoxicity caused by methylmercury. The elevated methylmercury sensitivity by Meh1 knockout almost completely disappeared in the presence of 3-methyladenine. CONCLUSIONS: Akr1 reduces methylmercury toxicity through palmitoylation of Meh1. Furthermore, the EGO complex including Meh1 reduces methylmercury toxicity by suppressing the induction of vacuole deformation caused by methylmercury. GENERAL SIGNIFICANCE: These findings propose that Meh1 palmitoylated by Akr1 may act as a constituent of the EGO complex when contributing to the decreased cytotoxicity by negatively controlling the induction of autophagy by methylmercury.

Immature Core protein of hepatitis C virus induces an unfolded protein response through inhibition of ERAD-L in a yeast model system.

The structural protein Core of hepatitis C virus (HCV), a cytosolic protein, induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in hepatocytes, and is responsible for the pathogenesis of persistent HCV infection. Using yeast as a model system, we evaluated mechanisms underlying Core-induced interference of ER homeostasis and UPR, and found that UPR is induced by the immature Core (aa 1-191, Core191) but not by the mature Core (aa 1-177, Core177). Interestingly, Core191 inhibits both ERAD-L, a degradation system responsible for misfolded/unfolded proteins in the ER lumen, and ERAD-M, a degradation system responsible for proteins carrying a misfolded/unfolded region in the ER membrane. In contrast, Core177 inhibits ERAD-M but not ERAD-L. In addition, requirement of an unfolded protein sensor in the ER lumen suggested that inhibition of ERAD-L is probably responsible for Core191-dependent UPR activation. These results implicate inadequate maturation of Core as a trigger for induction of ER stress and UPR.

History and development of The Journal of Toxicological Sciences.

The Journal of Toxicological Sciences, published by The Japanese Society of Toxicology (JSOT), is an international scientific journal covering the entire field of toxicology. This article reviews the history of The Journal of Toxicological Sciences as well as actions taken by the Editorial Committee to improve the journal and the results of these initiatives.

Methylmercury induces the expression of TNF-α selectively in the brain of mice.

Methylmercury selectively damages the central nervous system (CNS). The tumor necrosis factor (TNF) superfamily includes representative cytokines that participate in the inflammatory response as well as cell survival, and apoptosis. In this study, we found that administration of methylmercury selectively induced TNF-α expression in the brain of mice. Although the accumulated mercury concentration in the liver and kidneys was greater than in the brain, TNF-α expression was induced to a greater extent in brain. Thus, it is possible that there may exist a selective mechanism by which methylmercury induces TNF-α expression in the brain. We also found that TNF-α expression was induced by methylmercury in C17.2 cells (mouse neural stem cells) and NF-κB may participate as a transcription factor in that induction. Further, we showed that the addition of TNF-α antagonist (WP9QY) reduced the toxicity of methylmercury to C17.2 cells. In contrast, the addition of recombinant TNF-α to the culture medium decreased the cell viability. We suggest that TNF-α may play a part in the selective damage of the CNS by methylmercury. Furthermore, our results indicate that the higher TNF-α expression induced by methylmercury maybe the cause of cell death, as TNF-α binds to its receptor after being released extracellularly.

Adverse health effects of humidifier disinfectants in Korea: lung toxicity of polyhexamethylene guanidine phosphate.

Exposure to humidifier disinfectants was identified in 2011 as the potential cause of an outbreak of lung disease in Korea. It is estimated that over 8 million people have been exposed to humidifier disinfectants-chemicals added to the water used in humidifiers to prevent the growth of microorganisms-since their commercial introduction. The primary component of humidifier disinfectant products involved was polyhexamethylene guanidine phosphate (PHMG-P), a guanidine-based antimicrobial agent. Lesions observed in the lungs of patients were similar to those observed in laboratory animals exposed to PHMG-P. In this review, we outline the physicochemical and toxicological properties of PHMG-P, and introduce a putative mechanism for its lung toxicity based in large part on research findings to date.

Redox-dependent Regulation of Gluconeogenesis by a Novel Mechanism Mediated by a Peroxidatic Cysteine of Peroxiredoxin.

Peroxiredoxin is an abundant peroxidase, but its non-peroxidase function is also important. In this study, we discovered that Tsa1, a major peroxiredoxin of budding yeast cells, is required for the efficient flux of gluconeogenesis. We found that the suppression of pyruvate kinase (Pyk1) via the interaction with Tsa1 contributes in part to gluconeogenic enhancement. The physical interactions between Pyk1 and Tsa1 were augmented during the shift from glycolysis to gluconeogenesis. Intriguingly, a peroxidatic cysteine in the catalytic center of Tsa1 played an important role in the physical Tsa1-Pyk1 interactions. These interactions are enhanced by exogenous H2O2 and by endogenous reactive oxygen species, which is increased during gluconeogenesis. Only the peroxidatic cysteine, but no other catalytic cysteine of Tsa1, is required for efficient growth during the metabolic shift to obtain maximum yeast growth (biomass). This Tsa1 function is separable from the peroxidase function as an antioxidant. This is the first report to demonstrate that peroxiredoxin has a novel nonperoxidase function as a redox-dependent target modulator and that pyruvate kinase is modulated via an alternative mechanism.

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System.

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins.