Search

Reactive sulfur species (RSS) play a role in redox homeostasis; however, adaptive cell responses to excessive intracellular RSS are not well understood. Therefore, in this study, we generated transgenic (Tg) mice overexpressing cystathionine gamma-lyase (CSE) to produce excessive RSS. Contrary to expectations, tissue concentrations of RSS, such as cysteine persulfide (CysSSH), were comparable in both wild-type and CSE Tg mice, but the plasma concentrations of CysSSH were significantly higher in CSE Tg mice than in wild-type mice. This export of surplus intracellular RSS was also observed in primary hepatocytes of CSE Tg mice. Exposure of primary hepatocytes to the RSS generator sodium tetrasulfide (Na2S4) resulted in an initial increase in the intracellular concentration of RSS, which later returned to basal levels after export into the extracellular space. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during Na2S4 exposure, suggesting that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout and overexpression of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by Na2S4 exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity. These results suggest the presence of a cellular response to excess intracellular RSS that involves the extracellular efflux of excess CysSSH by a cystine-dependent transporter to maintain intracellular redox homeostasis.

Abstract Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)–Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ–S–1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN–Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

Methylmercury (MeHg) is a potent neurotoxin that affects particularly the developing brain. Since MeHg is a potent electrophilic agent, a wide range of intracellular effects occur in response to its exposure. Yet, the molecular mechanisms associated with MeHg-induced cell toxicity have yet to be fully understood. Activation of cell defense mechanisms in response to metal exposure, including the up-regulation of Nrf2- (nuclear factor erythroid 2-related factor 2)-related genes has been previously shown. Nrf2 is a key regulator of cellular defenses against oxidative, electrophilic and environmental stress, regulating the expression of antioxidant proteins, phase-II xenobiotic detoxifying enzymes as well phase-III xenobiotic transporters. Analogous to other electrophiles, MeHg activates Nrf2 through modification of its repressor Keap1 (Kelch-like ECH-associated protein 1). However, recent findings have also revealed that Keap1-independent signal pathways might contribute to MeHg-induced Nrf2 activation and cytoprotective responses against MeHg exposure. These include, Akt phosphorylation (Akt/GSK-3β/Fyn-mediated Nrf2 activation pathway), activation of the PTEN/Akt/CREB pathway and MAPK-induced autophagy and p62 expression. In this review, we summarize the state-of-the-art knowledge regarding Nrf2 up-regulation in response to MeHg exposure, highlighting the modulation of signaling pathways related to Nrf2 activation. The study of these mechanisms is important in evaluating MeHg toxicity in humans, and can contribute to the identification of the molecular mechanisms associated with MeHg exposure.

Methylmercury (MeHg), an environmentally toxic substance, causes site-specific neuronal cell death; while MeHg exposure causes death in cerebrocortical neurons, interestingly, it does not in hippocampal neurons, which are generally considered to be vulnerable to toxic substances. This phenomenon of site-specific neuronal cell death can be reproduced in animal experiments; however, the mechanism underlying the resistance of hippocampal neurons to MeHg toxicity has not been clarified. In this study, we comparatively analyzed the response to MeHg exposure in terms of viability and the expression characteristics of primary cultured cerebrocortical neurons and hippocampal neurons derived from fetal rat brain. Neuronal differentiated hippocampal neurons were more resistant to MeHg toxicity than cerebrocortical neurons, as indicated by a 2-3 fold higher half-maximal inhibitory concentration (IC50; 3.3 µM vs. 1.2 µM), despite similar intracellular mercury concentrations in both neuronal cell types. Comprehensive RNA sequencing-based gene expression analysis of non-MeHg-exposed cells revealed that 80 out of 15,208 genes showed at least 10-fold higher expression in hippocampal neurons than in cerebrocortical neurons, whereas six genes showed at least 10-fold higher expression in cerebrocortical neurons than in hippocampal neurons. In particular, genes related to neuronal function, including those encoding transthyretin and brain-derived neurotrophic factor, showed approximately 50-fold higher expression in hippocampal neurons than in cerebrocortical neurons. In conclusion, the resistance of hippocampal neurons to MeHg toxicity may be related to the high expression of neuronal function-related proteins. [ABSTRACT FROM AUTHOR]

Brain susceptibility to methylmercury (MeHg) is developmentally and regionally specific in both humans and rodents, but the mechanism is not well clarified. Reactive sulfur species (RSS) with high nucleophilicity can react with MeHg, leading to the formation of a less toxic metabolite bismethylmercury sulfide, thus exerting cytoprotection. In this study, we assessed the variation of RSS content in the rat brain and evaluated its relevance in sensitivity to MeHg. Analyses of fetal/juvenile rat brains showed low RSS levels in early developmental stages. Site-specific analysis of adult rat brains revealed that cerebellar RSS levels were lower than those of the hippocampus. Microscopically, RSS levels of the granular cell layer were lower than those of the molecular layer in the cerebellum. Thus, low RSS levels corresponded with age and site of the brain that is vulnerable to MeHg. Taken together with the finding that brain RSS were consumed during MeHg exposure, these results indicate that RSS is a factor that defines the specificity of MeHg vulnerability in the brain. [ABSTRACT FROM AUTHOR]

BACKGROUND: Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) plays a key role in detoxification of electrophiles via formation of glutathione (GSH) adducts and subsequent excretion into extracellular spaces. We found that reactive sulfur species (RSS), such as cysteine persulfides produced by cystathionine c-lyase (CSE), capture environmental electrophiles through formation of sulfur adducts. However, contributions of Nrf2 and CSE to the blockage of environmental electrophile-mediated toxicity remain to be evaluated. OBJECTIVES: The aim of this study was to clarify roles that CSE and Nrf2 play in the protection against various environmental electrophiles. We also wished to clarify the molecular basis of the developmental window of toxicity through investigating expression levels of Nrf2, RSS-producing enzymes, and sulfur nucleophiles during developmental stages of mice. METHODS: Wild-type (WT), CSE knockout (KO), Nrf2 KO, Nrf2/CSE double KO (DKO) mice, and their primary hepatocytes were analyzed in this study. Cadmium (Cd), methylmercury (MeHg), 1,4-naphthoquinone, crotonaldehyde, and acrylamide were used. We conducted Western blotting, realtime polymerase chain reaction (PCR), 3-(4,5-dimethylthiazol-2-yl)-2,5-triphenyl tetrazolium bromide (MTT) assays, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis, alanine transaminase (ALT) activity, histopathological analysis, and rotarod test. RESULTS: Primary hepatocytes from DKO mice were significantly more sensitive to the environmental electrophiles than each single KO counterpart. Both Nrf2 and CSE single KO mice were highly susceptible to Cd and MeHg, and such sensitivity was further exacerbated in the DKO mice. Lowerlevel expressions of CSE and sulfur nucleophiles than those in adult mice were observed in a window of developmental stage. CONCLUSIONS: Our mouse model provided new insights into the response to environmental electrophiles; while Nrf2 is recognized as a key transcription factor for detoxification of environmental electrophiles, CSE is crucial factor to repress their toxicity in a parallel mode. In addition, the sensitivity of fetuses to MeHg appears to be, at least in part, associated with the restricted production of RSS due to low-level expression of CSE. [ABSTRACT FROM AUTHOR]