Oxidative Stress in Methylmercury-Induced Cell Toxicity

Methylmercury (MeHg) is a hazardous environmental pollutant, which elicits significant toxicity in humans. The accumulation of MeHg through the daily consumption of large predatory fish poses potential health risks, and the central nervous system (CNS) is the primary target of toxicity. Despite well-described neurobehavioral effects (i.e., motor impairment), the mechanisms of MeHg-induced toxicity are not completely understood. However, several lines of evidence point out the oxidative stress as an important molecular mechanism in MeHg-induced intoxication. Indeed, MeHg is a soft electrophile that preferentially interacts with nucleophilic groups (mainly thiols and selenols) from proteins and low-molecular-weight molecules. Such interaction contributes to the occurrence of oxidative stress, which can produce damage by several interacting mechanisms, impairing the function of various molecules (i.e., proteins, lipids, and nucleic acids), potentially resulting in modulation of different cellular signal transduction pathways. This review summarizes the general aspects regarding the interaction between MeHg with regulators of the antioxidant response system that are rich in thiol and selenol groups such as glutathione (GSH), and the selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). A particular attention is directed towards the role of the PI3K/Akt signaling pathway and the nuclear transcription factor NF-E2-related factor 2 (Nrf2) in MeHg-induced redox imbalance.


Introduction
Mercury (Hg) is a global pollutant ubiquitously present in the environment with a potential toxic effect in humans. Hg can be emitted to the atmosphere by natural sources (volcanoes and forest fire) or by anthropogenic sources (industrial activities, mining, and coal combustion), and deposits into

Thiols and Selenols Play Fundamental Roles in MeHg-Induced Toxicity
Hg compounds react specifically with sulfhydryls (-SH) groups of cellular proteins and nonprotein molecules, forming stable complexes with defined stoichiometry, -S-Hg-R. MeHg's affinity for the anionic form of thiol (-SH) groups is extremely high and responsible for most of its toxicological effects [45]. Indeed, in biological media, MeHg is always complexed to -SH-containing ligands [46][47][48][49][50] and the therapeutic agents effective in reducing its body-burden contain -SH groups [51]. In this regard, it is noteworthy that the MeHg distribution to the different organs, as well as its excretion and, consequently, toxicity are likely related to its interaction with -SH groups on various biomolecules. MeHg interacts with the -SH group from non-protein molecules like glutathione (GSH), and also binds to L-cysteine forming the MeHg-L-cysteine complex, which is taken up by cells from different tissues by molecular mimicry as a surrogate of methionine [52]. Particularly important, MeHg-L-cysteine conjugates serve as a substrate for the neutral amino acid transporter, LAT-1, which transports MeHg (complexed with L-cysteine) across membranes [53]. Complexes of MeHg-cysteine and MeHg-GSH have been identified in blood [46,47], and complexes with MeHg-GSH in brain [48], liver [49], and bile [50].
Selenium (Se) is an essential trace element that is an integrant part of several enzymes (selenoenzymes) in the form of selenocysteine (Sec) [54]. Selenoenzymes play critical roles in the maintenance of cellular homeostasis since the reactivity of the selenol group (-SeH) in Sec is high [55,56]. Since selenols chemically resemble thiols (-SH) it also serves as a soft ligand for complexation with soft metals [57]. Given that under physiological conditions, selenols have a lower pKa (around 5.3) when compared to thiols, the fully ionized isoform (selenolates, -Se − ) are predominant and also more susceptible to electrophilic reactions by mercurials [58]. Five glutathione peroxidases (GPx) and three thioredoxin reductases (TrxR) are among the well-known redox-active selenoenzymes, with a selenol group in their redox active sites [55,56].
As previously mentioned, MeHg's affinity for sulfhydryls and selenols interferes with several important regulators of the antioxidant response that are rich in thiol and selenol groups. In this regard, the thioredoxin (Trx) and glutathione GSH systems play important roles in maintaining the redox balance in the brain, a tissue that is prone to oxidative stress due to its high-energy demand [59]. Taking it into account, the following sections address the predominant molecules targeted by MeHg in biological systems, as well as the effects of MeHg on the regulation/modulation of the PI3K/Akt signaling pathway and Nrf2 transcription factor

Glutathione (GSH)
GSH is the most abundant low molecular weight thiol compound synthesized in cells, reaching the concentrations of 1-10 mM, and it is the major antioxidant and redox buffer in human cells. In fact, GSH serves as a reducing agent for ROS and other unstable molecules, in the reaction catalyzed by GPx [60]. Several aspects of MeHg-induced neurotoxicity have been ascribed to GSH depletion. MeHg is able to interact with GSH, leading to the formation of an excretable GS-MeHg complex [61]. This interaction decreases the levels of GSH and, consequently, the GSH:GSSG (disulfide-oxidized) ratio, which contributes to the occurrence of oxidative stress [33]. It is worth mentioning that the pKa of GSH is high at physiological pH conditions and little GSH is ionized. However, at physiological pH values, glutathione S-transferase (GST) effectively lowers the pKa of the cysteine thiol of GSH from 9.3 in solution to 6.5-7.4 at the active site of various GSTs, resulting in formation of the catalytically active nucleophilic thiolate anion (GS − ) [62,63].
MeHg has been shown to bind GSH, both in animal models and cell culture systems [64,65]. Indeed, several in vitro [66,67] and in vivo [12,68] evidences have shown that MeHg exposure causes GSH depletion. In addition, GSH and enzymes related to its synthesis are known important targets of MeHg-induced developmental neurotoxicity. For example, Stringari et al. have shown that prenatal exposure to MeHg disrupts the postnatal development of the GSH antioxidant system (GSH levels, GPx and glutathione reductase (GR) activities) in mouse brain. Furthermore, the authors demonstrated that the biochemical alterations endured even when mercury tissue levels decreased and became indistinguishable from those noted in pups born to control dams [68].
Epidemiological and animal studies have shown that MeHg causes loss of neuronal cells in specific brain regions including the visual cortex and the cerebellum [28], with massive loss of cerebellar granule cells (CGC) [69]. Interestingly, the increased sensitivity of CGC has been attributed to the relatively low GSH content of these cells, although it is not the only factor recognized [70][71][72]. In this regard, decreased GSH levels have been reported in the cerebral and cerebellar cortices of MeHg-exposed animals whose cortical mercury levels were in the low micromolar range [12,68]. In the mammalian cerebrum and cerebellum, the intracellular GSH concentrations are in the millimolar (mM) range, therefore the generation of ROS induced by MeHg might be related to GSH-independent mechanisms as well, in addition to its direct interaction with GSH [66,73].
GSH is synthesized by the sequential addition of cysteine to glutamate followed by the addition of glycine. In this sense, MeHg selectively inhibits astrocytic uptake systems for cystine and cysteine transport [74,75], compromising GSH synthesis and the CNS redox potential [76]. The MeHg-induced inhibition of cystine transport and astrocytic GSH production would ultimately lead to decreased neuronal GSH levels and increased glutamate toxicity. It is worth mentioning that one of the major mechanisms involved in MeHg-induced neurotoxicity is glutamate dyshomeostasis. Indeed, inhibition of vesicular glutamate uptake, increase in spontaneous release of glutamate from presynaptic terminals, and inhibition of glutamate uptake by astrocytes are crucial phenomena related to MeHg-mediated neurotoxicity [31,77,78].
Furthermore, studies on the toxicological relevance of MeHg-GSH interaction have demonstrated that strategies able to raise GSH levels are protective against MeHg-induced neurotoxicity [79,80]. For instance, increased ROS formation and depleted mono-and disulfide GSH were observed in neuronal, glial, and mixed cultures, and supplementation with exogenous GSH protected against the MeHg-induced neuronal death [81].

Selenoenzymes
Selenium plays a crucial role in antioxidant defense, as one Se atom is absolutely required at the active site of all selenoenzymes, such as GPx and TrxR, in the form of selenocystein [82]. GPx is an antioxidant enzyme that, in the presence of tripeptide GSH, adds two electrons to reduce H 2 O 2 and lipid peroxides to water and lipid alcohols, respectively, while simultaneously oxidizing GSH to glutathione disulfide. The GPx/GSH system is thought to be a major defense in low-level oxidative stress, and decreased GPx activity or GHS levels may lead to the absence of adequate H 2 O 2 and lipid peroxides detoxification, which may be converted to OH-radicals and lipid peroxyl radicals, respectively, by transition metals (Fe 2+ ) [83]. Thioredoxin (Trx) is essential for maintaining intracellular redox status. The expression of this small (12 kDa) ubiquitous thiol-active protein is induced by ROS and an elevated serum level may indicate a state of oxidative stress. In this regard, TrxR, a NADPH-dependent lipid hydroperoxide reductase, uses NADPH to maintain the levels of reduced Trx via a mechanism similar to that used by GR to maintain GSH levels, contributing to the maintenance of thiol redox homeostasis in proteins. Importantly, the inhibition of TrxR impairs the cyclical regeneration of Trx activity, as Trx remains in the oxidized state [84,85].
The activity of selenoenzymes, such as GPx, may be negatively affected by MeHg [38,41]. In addition, TxrR may be particularly sensitive to mercury compounds after both in vitro [86,87] and in vivo exposure [39,88]. Glutathione peroxidase isoform 1 (GPx1) is an initial molecular target in CGC exposed to nanomolar concentrations of MeHg (300 nM) for 4 days in vitro [37]. Interestingly, the authors have shown that GPx-1 inhibition occurred before any changes on potential targets that are normally affected by high-dose MeHg exposures. Moreover, Gpx1 overexpression was able to prevent MeHg-induced neuronal death in these cells [37], suggesting that selenoproteins may help to prevent oxidative stress induced when MeHg directly disrupts proteins involved in cellular redox system pathways. Of particular toxicological significance, the results obtained by Farina et al. 2009 [37] indicate the potential role of Gpx1 as a primary target of MeHg. In agreement, another study has demonstrated that MeHg decreased GPx activity in three different models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells [38]. The authors have concluded that the inhibition of this antioxidant enzyme may cause a significant increase of MeHg-induced impairment of cell viability and oxidative stress. Ruszkiewicz et al., 2016 have demonstrated that MeHg affects the antioxidant Trx and GSH systems in a sex-and structure-specific manner, and that these changes are not associated with altered mRNA expression, but rather posttranscriptional mechanisms [89]. Although the direct interaction of MeHg with the selenol group of the selenoproteins is an important molecular mechanism involved in the MeHg-induced cytotoxicity and decreased protein function [37,90], posttranscriptional events concerning mercury-selenium interaction seem to be also involved in the decreased activity of these selenoproteins [91,92].
Se deficiency has been reported to exacerbate the neurodevelopmental effects induced by MeHg [93,94]. In fact, maternal exposure to MeHg decreased Se concentration and impaired GPx activity in the neural tissue of the offspring, but not in the maternal neural tissue. In addition, MeHg exposure of Se-deficient perinatal mice resulted in retarded neurobehavioral development [94]. A relatively recent study corroborates the hypothesis that Se deficiency is a contributing factor to MeHg-induced toxicity. The authors have shown that selenoprotein genes from antioxidant pathways, such as members of the GPx and TrxR families, were exclusively downregulated by MeHg in whole zebrafish (Danio rerio) embryos, and then rescued by elevated Se levels [91]. These results suggest that Se can reduce the toxicity of MeHg, although the mechanisms behind this interaction have yet to be fully clarified. In addition, it has been shown that the selenocompounds diphenyl diselenide (PhSe) 2 and ebselen possess thiol peroxidase-like activity, and neuroprotective properties against MeHg-induced neurotoxicity in vivo and in vitro [95][96][97][98]. Using an in vitro experimental model of cultured human neuroblastoma cells (SH-SY5Y), a recent study has shown that MeHg was able to inhibit the activity of Gpx and TrxR, while coexposure to (PhSe) 2 and MeHg showed a protective effect on both the activity and expression of TrxR [99]. The authors have speculated that a direct interaction of MeHg with the selenol groups of these enzymes and/or a reduction in enzyme synthesis may possibly be related to the inhibition of TrxR and GPx activities [33].

MeHg and PI3K/Akt Signaling Pathway
The phosphoinositide-3 kinase (PI3K)/Akt signaling pathway has been extensively reviewed by several authors [100][101][102]. Briefly, activation of receptor tyrosine kinases (RTK) or G-protein-coupled receptors (GPCR) lead the recruitment of PI3K in the plasma membrane. Following its recruitment, PI3K is activated and phosphorylates phosphatidylinositol (4,5) biphosphate (PIP 2 ) to phosphatidylinositol (3,4 5)-triphosphate (PIP 3 ). PIP 3 recruits Akt to the plasmatic membrane, allowing its activation through phosphorylation on threonine 308 and serine 473 by phospho-inositide-dependent kinase-1 (PDK1) [103] and mammalian target of rapamycin complex 2 (mTORC2) [104], respectively. In addition, negative regulation of the pathway is mediated by phosphatase and tensin homolog (PTEN), which dephosphorylates PIP 3 to PIP 2 [105]. It is well known that the PI3K/Akt signaling pathway plays a key role in multiple cellular processes, including metabolism, cell survival, proliferation, and motility [102], to name a few. In this regard, the activation of the PI3K/Akt signaling pathway can culminate in the inhibition of apoptosis in several cellular models [106][107][108], whereas the inhibition of this pathway could be associated with decreased cell viability [109]. Furthermore, it has been shown that ROS can activate the PI3K/Akt signaling pathway and inhibit PTEN [110,111], thus, inducing the Akt activation, as shown in several cultured cell models, such as fibroblasts [112], vascular smooth muscle cells [113], and mesanglial cells [114]. Sonoda et al., 1999 have demonstrated that the activation of Akt by hydrogen peroxide (H 2 O 2 ) in a glioblastoma cell line was blocked by wortmannin, a PI3K inhibitor, culminating in increased cell death [115].
Moreover, MeHg can also modulate the PI3K/Akt signaling pathway. Indeed, exposure to 100 nM MeHg for 24 h induced apoptosis in differentiating PC12 cells, and decreased Akt phosphorylation [116]. Corroborating these findings, Pierozan et al., 2017 have also demonstrated that MeHg reduced neuronal viability and induced caspase 3-dependent apoptosis with downregulated PI3K/Akt pathway in primary cortical neurons treated with 1 µM MeHg for 24 h. In this study, the authors noted increased oxidative damage, suggesting that MeHg downregulated Akt phosphorylation despite the noted increase in ROS production [117]. A recent in vivo study using 30-day-old pups from pregnant rats treated with 2 mg/kg MeHg from gestational day 5 until parturition, has shown that the pups prenatally exposed to MeHg failed to increase hippocampal oxidative stress, while the levels of Akt phosphorylation were decreased. These results suggest that MeHg may regulate the Akt signaling pathway by a mechanism independent of ROS generation [9].
On the other hand, a submicromolar MeHg exposure (1 µmol/L) induced Akt activation, probably secondary to increased ROS production (in pancreatic β cell-derived HIT-T15 cells). Moreover, the antioxidant N-acetyl-L-cysteine (NAC) prevented MeHg-induced upregulation of Akt phosphorylation but did not reverse PI3K activity in these cells [118]. Similar results were obtained in isolated mouse pancreatic islets [118], suggesting that MeHg-induced oxidative stress may be involved in the regulation of Akt phosphorylation independently of PI3K in pancreatic cells. The same authors have shown similar results in mice treated for 2-4 weeks with 20 µg/kg MeHg, demonstrating an increase in oxidative stress parameters and upregulation of Akt phosphorylation in pancreatic islets [118]. Furthermore, a low-dose MeHg exposure (up to 2 µM) increased Akt phosphorylation, presumably by inactivation of PTEN through S-mercuration (in neuroblastoma SH-SY5Y cell line). Pretreatment with the PI3K/Akt inhibitor, wortmannin, enhanced MeHg-induced cytotoxicity [119]. In this study, the authors have suggested that low-dose MeHg exposures might be associated with activation of cell survival responses [120].

MeHg Regulation of Nrf2 Activity
As mentioned before, the increase in ROS generation and the disruption of the antioxidant defense system are primary mechanisms related to MeHg-induced cell toxicity and neurodegeneration [1,33]. In this sense, a recent body of evidence has suggested that MeHg can modulate the Nrf2, the transcription factor involved in the regulation of the antioxidant defense system. Nrf2 is a transcription factor that belongs to cap 'n' collar (CNC) family, with a basic leucine zipper in its structure. This protein is an important regulator of the antioxidant cell response, regulating the expression of around 1055 genes involved not only in the antioxidant defenses, but also in cell proliferation, metabolism, and immune and detoxifying responses. Once activated, Nrf2 is translocated to the cell nucleus and forms heterodimers with other transcript such as c-Jun and small Maf proteins, binding to the antioxidant response element (ARE: 5 -GTGACNNNGC-3 ), favoring the transcription of Nfr2-related genes [121][122][123]. The main regulator of Nrf2 activity is the Kelch-like ECH-associated protein 1 (Keap1) protein [123]. In basal conditions, Keap-1 is associated to Nrf2 inducing Nrf2 ubiquitination by E3 ubiquitin ligase, and consequent degradation through 26S proteasome. The Keap-1 structure contain some reactive cysteine residues (Cys151, Cys273, Cys288 and Cys297), which can be modified under oxidative stress conditions or by electrophilic compounds, promoting the disruption of the Keap-1/Nrf2 association and Nrf2 translocation to the cell nucleus, and consequent gene expression [121,123,124]. In addition, besides the negative regulation of Keap-1 on Nrf2 activity, the modulation of this protein by other signaling pathways has also been described. In fact, nuclear translocation of Nrf2 may involve the PKC and AMPK-dependent phosphorylation [125,126]. An association between PI3K/Akt activation and Nrf2 up-regulation has also been reported [127][128][129][130]. Once activated, Akt promotes the inhibition of GSK-3β through phosphorylation on ser-9. GSK-3β can induce Nrf2 phosphorylation, creating a degradation domain that is recognized by the ubiquitin ligase, targeting Nrf2 for proteasomal degradation [131][132][133][134]. GSK-3β can also phosphorylate a member of the src-kinase family named Fyn, which can phosphorylate Nrf2, favoring its export from the cell nucleus [130,135].
Since MeHg is a potent oxidative stress inducer and an electrophilic agent, the activation and up-regulation of Nrf2 have been described upon exposure to this metal. This hypothesis is supported by numerous data showing an increase in the Nrf2-related gene expression (such as: ho-1, nqo-1, cglc and Nrf2), and also an increase in the Nrf2 nuclear translocation, after exposures to MeHg in vitro [67,71,120,136] and in vivo [137]. Furthermore, it has been demonstrated that Nrf2 genetic knockdown can increase the susceptibility to MeHg toxicity, and treatment with compounds that are able to increase Nrf2 activity ameliorate some consequences of MeHg-induced toxicity [136,138]. In this sense, it seems that MeHg-induced Nrf2 activation is associated with a cellular defense mechanism in response to this metal exposure.
MeHg is able to disrupt the Nrf2/Keap-1 association, which may be associated with an increase in the Nrf2 activation [136,139]. It has been shown a direct interaction between MeHg and the cysteine residues in Keap-1 structure (Cys151, Cys368, and Cys489) [136,139,140]. As mentioned previously, MeHg can react with GSH to form a MeHg-SG adduct that is transferred to extracellular space by the multidrug-resistance associated protein (MRP) [1]. This GSH adduct readily undergoes S-transmercuration with cellular proteins, inducing the mercuration of Cys319 residue in the Keap-1 structure [141]. Cys151 residue is essential to electrophile-mediated disassociation of Keap1 from Nrf2, and The Cys319 residue is critical to the ubiquitin E3 ligase activity and consequent Nrf2 degradation [136,142,143]. In this sense, it is possible to speculate that the MeHg-induced modifications in these cysteine residues, through direct interaction with Cys151 and Cys319 or by ROS-mediated modifications, could disrupt the inhibitory effect of Keap-1 in Nrf2 activation, allowing its translocation to the cell nucleus. It is noteworthy that the cysteine residues in Keap-1 are also potential targets for oxidation mediated by ROS [124,130]. However, further studies are necessary to elucidate the role of ROS in MeHg-induced Nrf2 activation.
MeHg can induce the modulation of PI3k/Akt, and the inhibition of this signaling pathway was able to attenuate the MeHg-induced Nrf2 activation [71]. An increase in GSK-3β phosphorylation was also observed 6 h after an in vitro MeHg exposure in primary cortical astrocytes. MeHg decreased the expression of Fyn and Sp1 (the transcription factor associated to fyn expression), and Fyn nuclear localization, which may possibly suggest a Fyn downregulation in these cells [120]. Thus, it is speculated that MeHg promotes Akt activation and consequent inhibitory phosphorylation of GSK-3β. In these conditions, the downregulation of Fyn phosphorylation may inhibit the Fyn-mediated Nrf2 nuclear export, enhancing the Nrf2 nuclear localization. These authors have also shown that the increase in the Nrf2-related genes was followed by downregulation of Sp1-related genes (such as fyn and tgf-β1) [120]. This finding corroborates the idea that Sp1 can interact with Nrf2 at promoter sequences, suppressing the expression of Sp1 specific target genes [144].

Conclusions
MeHg is a hazardous environmental pollutant of great concern to public health because of its neurotoxic effects. Due to its electrophilic nature, MeHg can react with nucleophiles such as sulfhydryland selenol-containing proteins and low-molecular-weight molecules in biological systems. MeHg's affinity for the anionic form of thiol and selenol groups is extremely high and responsible for most of its toxicological effects. Indeed, MeHg can interfere with several crucial regulators of the antioxidant response that are rich in thiol and selenol groups such as glutathione (GSH), the major antioxidant and redox buffer in human cells, and the antioxidant selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). Consequently, the excessive generation of ROS is an important phenomena that culminates in cell death. In this regard, antioxidant molecules have been reported as important protective agents against MeHg-induced toxicity. Our understanding of the critical targets of MeHg is incomplete, and detailed experimental data regarding the mechanism of action are needed. For instance, given that MeHg is known to generate ROS, and that mammalian cells activate Nrf2-mediated transcription in response to ROS, it is not surprising that Nrf2 activation has been demonstrated in response to MeHg exposure. However, relatively little information has been found about the potential mechanisms involved in MeHg-induced Nrf2 activation. In addition, the relationship between the PI3K/Akt signaling pathway and MeHg toxicity is still very limited, and consideration should be given to future research. In conclusion, the data presented in this review suggest that multiple mechanisms are involved in MeHg-induced oxidative stress and cell toxicity. Taken together, the data highlight several promising directions for future research in this area.