Glutathione antioxidant system and methylmercury-induced neurotoxicity: an intriguing interplay

Abstract

Methylmercury (MeHg) is a toxic chemical compound naturally produced mainly in the aquatic environment through the methylation of inorganic mercury catalyzed by aquatic microorganisms. MeHg is biomagnified in the aquatic food chain and, consequently, piscivorous fish at the top of the food chain possess huge amounts of MeHg (at the ppm level). Some populations that have fish as main protein’s source can be exposed to exceedingly high levels of MeHg and develop signs of toxicity. MeHg is toxic to several organs, but the central nervous system (CNS) represents a preferential target, especially during development (prenatal and early postnatal periods). Though the biochemical events involved in MeHg-(neuro)toxicity are not yet entirely comprehended, a vast literature indicates that its pro-oxidative properties explain, at least partially, several of its neurotoxic effects. As result of its electrophilicity, MeHg interacts with (and oxidize) nucleophilic groups, such as thiols and selenols, present in proteins or low-molecular weight molecules. It is noteworthy that such interactions modify the redox state of these groups and, therefore, lead to oxidative stress and impaired function of several molecules, culminating in neurotoxicity. Among these molecules, glutathione (GSH; a major thiol antioxidant) and thiol- or selenol-containing enzymes belonging to the GSH antioxidant system represent key molecular targets involved in MeHg-neurotoxicity. In this review, we firstly present a general overview concerning the neurotoxicity of MeHg. Then, we present fundamental aspects of the GSH-antioxidant system, as well as the effects of MeHg on this system.

1. Introduction - Fundamental aspects concerning MeHg chemistry and toxicity

Mercury is a chemical element with symbol Hg, atomic number 80 and weight 200.59. Hg is not essential to living cells; conversely, it is highly toxic to animals (including humans) and plants [1]. Hg is present in the environment mainly in three chemical forms: elemental Hg vapor, inorganic Hg salts, and organic Hg compounds, such as methylmercury (MeHg) [2]. MeHg is naturally produced mainly in the aquatic environment by the methylation of inorganic mercury in a process that takes place in aquatic microorganisms [3]. In the aquatic milieu, MeHg is bioaccumulated through the aquatic food chain, reaching concentrations over 1 ppm in certain fish species [4]. Seafood ingestion is the most form of human exposure to MeHg. Accordingly, populations that have fish as main protein’s source are exposed to toxic MeHg levels [1].

Once in the body, MeHg can be toxic to several organs/systems, such as the kidney [5,6], the cardiovascular system [7,8], the immune system [9] and the reproductive system [10,11]. However, the central nervous system (CNS) represents a main target for MeHg-toxicity. In this regard, a vast epidemiological literature has reported impressive neurotoxic symptoms in humans highly exposed to MeHg, which include but are not limited to somatosensory disturbances and psychiatric symptomatology (for a review, see reference [12]). A critical event related to MeHg-neurotoxicity is its transplacental transport, allowing for the targeting of the fetus brain [13]. In line with this, epidemiological evidence shows that the exposure of pregnant women to MeHg may cause neurological deficits in their children [14,15]. Of note, developmental MeHg exposure at levels that commonly do not affect the mature (mother’s] brain is responsible for brain injury in the developing one [16].

Concerning molecular mechanisms related to MeHg-induced toxicity, it is important to mention that MeHg (CH₃Hg⁺) has a reactive center with one positive charge in the Hg atom. Therefore, it has a relative high affinity for electrons [17], which is the cause of its relative high reactivity with nucleophilic centers, i.e. selenohydryl (-SeH or selenol) or sulfhydryl (-SH or thiol] groups. Thiols can be found in either low molecular mass molecules (i.e. glutathione (GSH]] or in proteins [18]. In eukaryotic cells, selenols are primarily present in a small number of proteins (selenoproteins] as selenocysteine [19]. In the human genome, 25 genes for selenoproteins have been identified and some of them are oxidoreductases that catalyze reactions of the detoxification of oxidants, such as glutathione peroxidase and thioredoxin reductase [20].

MeHg-induced neurotoxicity is notably linked to its interaction with either thiols or selenols groups. Such interaction changes the structure (and sometimes the function] of several proteins, as well as may change the redox state of low-molecular antioxidants, contributing to MeHg-induced oxidative stress [21]. Mechanisms other than oxidative stress are also involved in MeHg-induced neurotoxicity. In fact, changes in intracellular calcium homeostasis [22] and in proper functioning of the glutamatergic neurotransmitter system [23] also represent central events in MeHg-induced neurotoxicity. In this regard, there seems to be a relationship between the pro-oxidant properties of MeHg with its effects on calcium and glutamatergic system. In fact, it has been reported that increased levels of hydroperoxide, which can represent a consequence of MeHg-induced inhibition of glutathione peroxidase (discussed latter, [24]), are able to decrease astrocyte glutamate uptake [25], which may culminate an excitotoxicity and calcium-mediated neuronal damage [23, 26]. Accordingly, the oxidation of sulfhydryl-containing proteins may affect chemical neurotransmition through modulation of neurotransmitters release [27], as well as their binding to specific receptors [28]. In this regard, there is a significant number of nucleophilic molecules that have been reported to be targets mediating MeHg-induced toxicity (i.e., thioredoxin reductase [29,30], selenoprotein W [31], glucose-6-phosphate dehydrogenase [32,33], and 5’-deiodinase [13]). The direct interaction of the MeHg’s mercury atom with thiol or selenol groups of these target proteins has been proposed as a mechanism of toxicity [34, 35, 36]. Although any thiol or selenol-containing molecule could represent a potential target for MeHg, it is important to mention that selenols are more nucleophilic than thiols [37], consistent with the fact that selenoproteins represent primary/preferential targets compared to sulfhydryl proteins [24]. It is expected that MeHg will react preferentially with the GPx’s selenocysteine residue than with the GSH’s cysteine residue when all components (MeHg, GPx and GSH) are at equimolar concentrations. However, the intracellular levels of GSH are significantly higher (at the mM range) than those of GPx in the biological systems, which will also dictate the hierarchy of MeHg’s interaction toward components of the GSH antioxidant system. The hierarchical profile of MeHg interaction with the selenol groups of different selenoproteins will depend on their pKa values; anyway, comparative studies concerning the MeHg’s affinity for different selenoproteins (i.e. glutathione peroxidase and thioredoxin reductase) have yet to be addressed. Even though MeHg can target diverse nucleophilic molecules, this review will focus on the glutathione (GSH) antioxidant system. The choice of delving into this specific system is not only related to the fact that several toxic effects resulting from MeHg exposure are due to its effects toward the GSH and related enzymes (discussed later), but also because the GSH antioxidant system significantly affects MeHg kinetics and toxicity.

2. The glutathione antioxidant system

The term glutathione antioxidant system, used in most of the literature and in this review article, depicts the low-molecular weight glutathione (GSH) and the main enzymes involved in its metabolism. The tripeptide GSH (γ-l-glutamyl-l-cysteinylglycine) is the most important low-molecular sulfhydryl antioxidant, which is present intracellularly in millimolar concentrations. Its most important functions, which are particularly dependent on the thiol group of its cysteinyl residue, are related to the detoxification of either xenobiotics or endogenous oxidants [38]. Taking into account the great number of potentially deleterious molecules that can be detoxified in a GSH-dependent manner [39], it is predictable that impaired GSH homeostasis has been associated with human pathological conditions [40], including those related to the CNS [41].

Accordingly, prior to considerations on the role of this important antioxidant system in the detoxification and/or metabolism of pro-oxidant molecules, it is important to mention that the proper maintenance of a physiological level of oxidant challenge, currently termed oxidative eustress, is essential for governing life processes through redox signaling [42, 43]. Thus, oxidants such as hydrogen peroxide, whose levels may be increased after MeHg exposure (discussed latter), will play significant deleterious effects in oxidizable biomolecules when the physiological oxidant challenge (eustress) is surpassed, which may culminate in oxidative damage (distress). Conversely, at physiological levels, hydrogen peroxide displays essential roles in regulating cell signaling and metabolism through the oxidation of proteic nucleophilic groups, which operate as redox switches [44]. In this regard, the GSH antioxidant system may play an important role in redox signaling either via glutathionylation (direct modification of protein cysteine residues by the addition of GSH) or via metabolism of hydrogen peroxide, detailed in item 2.3.

2.1. GSH synthesis

GSH is synthesized in two ATP-consuming reactions that take place in the cytosol (Figure 1A, events a and b). Initially, the enzyme glutamate cysteine ligase (GCL) catalyzes the condensation of cysteine and glutamate to form the dipeptide γ-glutamylcysteine (γGluCys). In another reaction, glycine is added to form the tripeptide in a reaction catalyzed by GSH synthetase. The first reaction, catalyzed by GCL, represents a regulated step that controls GSH synthesis. Chemical inhibition of GCL with the buthionine sulfoximine (BSO) has been reported to significantly decrease GSH levels in both in vitro [45] and in vivo [46] conditions. Of note, both the GCL and GSH synthase genes are regulated by the nuclear transcription factor erythroid-2-related factor 2 (Nrf2) [47], which translocates into the nucleous during oxidative stress, mainly via oxidation of an associated protein (the Kelch-like ECH-associated protein 1; KEAP1) [48]. Nuclear translocation of Nrf2 stimulates the transcription of Nfr2-related genes, such as phase 2 detoxifying and antioxidant enzymes (i.e., GCL). Not only oxidative stress, but also additional events (i.e. PKC and AMPK-dependent phosphorylation) can stimulate Nrf2 translocation to the nucleus [49,50], increasing antioxidant and xenobiotic-metabolizing capacity.

Figure 1: General overview of glutathione synthesis and most important intracellular roles.

A) Glutathione synthesis - The tripeptide glutathione is synthesized in two ATP-consuming reactions. Event (a) represents the condensation of cysteine and glutamate to form the dipeptide γ-glutamylcysteine in a reaction catalyzed by glutamate cysteine ligase. Event (b) represents the addition of glycine to form the tripeptide in a reaction catalyzed by GSH synthetase. B) GSH-GSSG redox cycling - Glutathione peroxidases catalyze the reduction of H₂O₂ and organic peroxides at the expenses of electrons from GSH (event a). The oxidized glutathione generated by the GPx-catalyzed reaction can be reduced by glutathione reductase (event b), which uses NADPH generated mainly by the oxidative phase of the pentose phosphate pathway (event c). C) GSH complexation with xenobiotics - The conjugation of glutathione with diverse xenobiotics is catalyzed by glutathione-S-transferases (event a), forming a xenobiotic-GSH conjugate that can be exported from the intra- to the extracellular environment via multidrug resistance-associated proteins (event b). D) Thiol oxidation - The formation of protein disulfides can occur via either the oxidation of two protein thiols (from a single or different proteins, events a and b, respectively) or when a single sulfhydryl protein forms a disulfide with GSH via glutathionylation, forming a mixed disulfide (event c). E) Glutaredoxins - Glutaredoxins use electrons from glutathione to reduce oxidized target proteins, restoring the reduced state in cysteine(s) from proteins (event a) or from a mixed disulfide (event b).

GSH (reduced glutathione = γ-l-glutamyl-l-cysteinylglycine); GSSG (oxidized glutathione); Glu (glutamate); Cys (cysteine); γGluCys (dipeptide γ-glutamylcysteine); GCL (glutamate cysteine ligase); GS (GSH synthetase); GPx (glutathione peroxidase); GR (glutathione reductase); GSTs (glutethione-S-transferases); RX (xenobiotic); ROOH (peroxide = hydrogen or organic peroxide); Grx (glutaredoxin).

With respect to the regulation of GSH synthesis, is important to mention that the maintenance of proper intracellular GSH concentrations is achieved through tightly regulated events, which occur at both transcriptional and post-transcriptional (either mRNA stabilization/destabilization or post-translational modification) levels. The major determinants of GSH synthesis are the availability of precursor (cysteine is a main limiting amino acid), as well as the activity/levels of GCL and GS [47]. The expression of GSH synthetic enzymes is increased during oxidative stress and the main transcription factors involved in this process include Nrf2/Nrf1 via the antioxidant response element (ARE), activator protein-1 (AP-1) and nuclear factor κ B (NFκB) [47]. Nrf2 seems to play a pivotal role in regulating GSH levels.

Nrf2 is a transcription factor of the cap ‘n’ collar-basic leucine zipper proteins (CNC-bZIP) [51–53]. It is a key regulator of the antioxidant cell response, modulating the expression of more than a thousand genes involved in the antioxidant defenses, metabolism, cell proliferation, and immune and detoxifying responses, including GCL and GS. When activated, Nrf2 translocates to the nucleus and forms heterodimers with other transcripts (i.e. c-Jun and small Maf proteins), binding to the antioxidant response element (ARE), favoring the transcription of specific genes [54]. Keap1 (Kelch-like ECH-associated protein 1) is the most important regulator of Nrf2 activity. Under basal conditions, Keap-1 interacts with Nrf2, stimulating its ubiquitination and consequent proteolysis. A well-known mechanism associated with the disruption of the Keap-1/Nrf2 association is the oxidation of specific thiol groups from Keap-1 (Cysl51, Cys273, Cys288 and Cys297], which leads to Nrf2 translocation to the nucleus, stimulating the expression of several genes [55]. Additional mechanisms modulating Nrf2 nuclear translocation and/or degradation involve protein phosphorylation. GSK-3β, by stimulating Nrf2 phosphorylation, creates a degradation domain that, once recognized by ubiquitin ligase, triggers Nrf2 proteasomal degradation [56]. Additionally, GSK-3β phosphorylates Fyn (a member of the src-kinase family], which can phosphorylate Nrf2, leading to nuclear export ubiquitination, and degradation of Nrf2 [57]. In this scenario, PI3K/Akt activation inhibits (by phosphorylation] GSK-3p, thus preventing Nrf2 degradation. Additionally, PI3K/Akt induces extracellular-signal related kinase (Erk1/2] activation, which favors Nrf2 translocation to the nucleus [58].

Because Nrf2 activation results in the upregulation of several antioxidant enzymes and cy to protective genes, several lines of evidence have pointed it as an attractive therapeutic target for oxidative stress-related pathological conditions [59,60], including neurodegenerative diseases [61]. As result of the stimulation of Nrf2 translocation after the oxidation of critical cysteine residues, several electrophiles have been reported to display protective effects in a Nrf2-dependent manner (for review, see [61]]. In addition to GCL and GS (involved in GSH synthesis], further proteins linked to the GSH system are also upregulated via Nrf2, such as GR [62] and some members of the GST [63] and GPx [64] families. Of note, the neurotoxicant MeHg has been reported to stimulate Nrf2 activation and, in turn, Nrf2 has been reported to protect against MeHg toxicity (discussed latter in Section 3.6).

2.2. Glutathione S-transferase: GSH complexation with xenobiotics

Glutathione S-transferase (GST), which are present in bacteria, plants, and animals, is also part of the GSH system and represents a large family of proteins that act in detoxication pathways by catalyzing the conjugation of GSH with diverse xenobiotics (Figure 1C, event a), usually resulting in more efficient transport and elimination [65]. They also participate in a number of anabolic and catabolic pathways and play an important role in defense against oxidative stress by reducing peroxides and dehydroascorbates [66]. Human GSTs are divided into eight classes (Alpha, Mu, Pi, Theta, Kappa, Zeta, Omega, and Sigma), which are based on protein structure similarity, as well as kinetic properties [67, 68].

From a biochemical point of view, it is noteworthy that the basis for all GST catalytic activities relies on their capability of lowering the pK_a of the thiol group of GSH from 9.0 (in aqueous solution) to approximately 6.5, when GSH is bound in the enzyme’s catalytic site [69]. With the reduction of the thiol’s pK_a, GSH will predominantly be as the thiolate (GS-) anion and, consequently, will be more reactive to electrophilic xenobiotics. The binding of these compounds to the hydrophobic motif of GSTs also favors the occurrence of the reaction [70]. The conjugation of GSH with the diverse xenobiotics leads to the formation of a conjugate/complex (xenobiotic-GSH) that is generally more hydrophilic than the xenobiotic alone, allowing for export and/or excretion. The export of the xenobiotic-GSH conjugate from the intra- to the extracellular environment generally occurs via a transport protein from the multidrug resistance-associated proteins family [71], (Figure 1C, event b). GSTs are involved in the detoxification of several xenobiotics (i.e., polychlorinated biphenyls, triazines, thiocarbamates, sulfonylureas, chloracetanilides, just to name a few), but also catalyze the conjugation of GSH with endogenous compounds (i.e., 4-hydroxinonenal, formed as a product of lipid peroxidation) [72]. Of particular importance, some lines of evidence suggest that GSTs can moderate MeHg transport and toxicity [73], suggesting its participation in the catalysis of the complexation of GSH with MeHg; this is discussed in item 3. Even though GSH conjugation has been identified as an important detoxication process, several GST-dependent bioactivation reactions have been identified [74].

2.3. Glutathione peroxidase and glutathione reductase: GSH-GSSG redox cycling

As already mentioned, GSH contributes to the detoxification of endogenous oxidants. In this regard, GSH is able to directly (non-enzymatically) react with hydroxyl (⁻OH) and superoxide (O₂₋) radicals. Moreover, GSH donates electrons for the detoxification (reduction) of H₂O₂ and organic peroxides in a reaction catalyzed by GSH-dependent oxidoreductases - enzymes of the glutathione peroxidase (GPx) family. Glutathione peroxidases (GPxs) represent an important family of antioxidant enzymes that catalyze the reduction of H₂O₂ and organic peroxides at the expenses of electrons from GSH (Figure 1B, event a) [75]. In humans, 8 isoforms (GPx1 - GPx8) have been so far identified; among then, the cytosolic (GPx1), the gastrointestinal-specific (GPx2), the plasma (GPx3) and the phospholipid hydroperoxide (GPx4) contain selenocysteine in the catalytic site. GPx6, which is expressed in the olfactory epithelium, is a seleneprotein just in humans, but not in rodents and other species [76]. GPx5, GPx7 and GPx8 contain cysteine in the active site [75]. The presence of critical nucleophilic groups (selenols or thiols) at their active sites implies that all GPx isoforms can be target of electrophiles, such as MeHg (discussed latter).

In addition to alcohols or water, derived from the reduction of organic peroxides or H₂O₂, respectively, the usual GPx-catalyzed reaction also produces oxidized glutathione (GSSG), which is a disulfide formed from the oxidation of two GSH molecules. The GSSG generated by the GPx-catalyzed reaction can be reduced by the flavoenzyme glutathione reductase (GR) (Figure 1B, event b), which uses NADPH as reducing agent [77]. The reduction of GSSG to GSH (2 molecules) is needed to the maintenance of the proper GSH-GSSG redox cycling; the availability of NADPH, generated mainly by the oxidative phase of the pentose phosphate pathway (PPP) (Figure 1B, event c) represents a crucial event [78]. During oxidative stress, GSSG can oxidize critical protein thiols through the formation of mixed protein-GSH disulfides (Figure 1B, event d), affecting protein function. This process can be mitigated by the export of GSSG from the cell by multidrug resistance protein 1 [79], but it results in decreased intracellular GSH levels.

As already mentioned, the detoxification of peroxides and the maintenance of the proper GSH-GSSG redox cycling are crucial events to avoid extreme oxidative events toward lipids peroxidation, protein and nucleic acids. In this scenario, GPx and GR are two important enzymes of the GSH antioxidant system that, with the reducing support from GSH, help to maintain appropriate thiol redox state, preventing such oxidative events. Figure 1B depicts the main events related to the GSH-GSSG redox cycling.

2.4. Glutaredoxins

In sulfhydryl-containing proteins, their thiol groups (PSH] may display crucial roles in modulating their respective functions [80]. As consequence, pro-oxidative conditions that cause excessive PSH oxidation may affect protein functions and, consequently, lead to pathological conditions. Particularly in the CNS, oxidation of PSH may affect neurotransmitter release [27] and their binding to specific receptors [28], which importantly affects chemical neurotransmission.

Depending on the oxidative stimulus, PSH oxidation can lead to the formation of disulfide linkages (–S–S–), cysteinyl radical (P-S⁻], sulfenic (PSOH), sulfinic (PSO₂H), sulfonic (PSO₃H) acids, etc. [81]. Of note, PSH oxidation to disulfides is frequently reversible in cells under normal physiological conditions [82]; disulfides can be formed by either the oxidation of two PSHs (from a single or different proteins, Figure 1D, events a and b, respectively] or when a single PSH form a disulfide with GSH (or free cysteine] via glutathionylation (or cysteinylation], respectively, forming a mixed disulfide (Figure 1D, event c]. The reduction of disulfides (in proteins or mixed disulfides] is done at the expense of electrons initially derived from NADPH in events that may have glutaredoxins or thioredoxins as intermediary reducing agents [83]. Because thioredoxin and thioredoxin reductase are not part of the GSH system, these proteins will be not discussed in this review (for details about the role of thioredoxin and thioredoxin reductase in reducing oxidized sulfhydryl proteins, see Lu and Holmgren [84]).

Glutaredoxins (Grxs) represent a group of sulfhydryl-containing proteins that can be considered part of the GSH system. Depending on the number of active site cysteine residues, they are divided into dithiol and monothiol Grxs [85]. In dithiol Grxs, cysteine residues at the active centre may be in either a reduced (thiol) or an oxidized (disulfide) form. In the oxidized form (disulfide), these Grxs can be non-enzymatically reduced by GSH. Once reduced, Grxs act in a system in which electrons are transferred to oxidized target proteins, restoring the reduced state in cysteine(s) from proteins (Figure 1E, event a) or from a mixed disulfide (Figure 1E, event b). Consequently, dithiol Grxs display a crucial role in post-translational redox modification, which have been implied in the modulation of numerous physiological and disease-related conditions, such as immune defense, cardiac hypertrophy, hypoxia-reoxygenation insult, neurodegeneration and cancer [86]. Figure 1E depicts the main events related to the reduction of oxidized sulfhydryl proteins and mixed disulfides by dithiol Grxs. In contrast to dithiol Grxs, monothiol Grxs function mainly in iron homeostasis and in the biosynthesis of FeS proteins [87].

3. The complex interplay between MeHg and the GSH antioxidant system: protective and toxic consequences

3.1. MeHg and GSH interaction

As already mentioned, the interaction of MeHg with nucleophilic groups (i.e., thiols) has been reported as a key event mediating its toxicity. In this scenario, more than 4 decades have passed since the direct chemical interaction between MeHg and GSH was reported in the literature. Such interaction displays a crucial role in decreasing MeHg deposition in tissues [88] and increasing Hg excretion in the bile of MeHg-exposed rats [89], indicating a protective role of GSH against MeHg-toxicity. From the molecular point of view, it is noteworthy MeHg can interact with the thiol group of GSH forming a complex (MeHg-GSH) (Figure 2, event a), which can be exported from the cells (Figure 2, event b). In line with this, in vitro experiments with cultured cells have shown that (i) increased GSH levels are protective against MeHg-induced neurotoxicity [90,91] and (ii) GSH monoethyl ester (a GSH precursor) protected against MeHg-neurotoxicity in a manner dependent on MRP1-mediated efflux [92], highlighting the important role of this family of proteins (multidrug resistance-associated proteins) in exporting the MeHg-GSH complex from cells. Under in vivo conditions, treatment with N-acetylcysteine (NAC, a precursor of cysteine, essential for GSH synthesis) reduced MeHg levels in the developing embryo in a rodent model [93]. The authors observed that NAC increased the urinary excretion of MeHg, highlighting its potential role as antidote agent in MeHg poisonings. Consistent with this, Dutczak and Ballatori [94] reported that the MeHg-GSH complex represents a major form by which MeHg is transported across liver canalicular membranes into bile, a major route of excretion. According to these observations, GSH depletion has been reported as a consequence of MeHg exposure in rodent models [95,96]. Even though the formation of the MeHg-GSH complex tend to decrease MeHg toxicity, it is important to mention that MeHg can be exchanged between different thiols, allowing for the transference of MeHg from the MeHg-GSH complex to another thiol (i.e., sulfhydryl protein) [97], leading to PSH oxidation and potential changes in protein function (Figure 2, event c). Thus, even though MeHg is much more reactive toward thiols than MeHg-GSH, one may hypothesize that this complex could also contribute to thiol oxidation because, according to the studies of Rabenstein and collaborators [97], MeHg can be exchanged between different thiols. It is noteworthy that MeHg can also interact directly with free cysteine [98], forming a cysteine-MeHg complex, which can be taken up by cells via a neutral amino acid carrier transport System L [99]. Even though the formation of this cysteine-MeHg complex can theoretically decrease cysteine availability for GSH synthesis, to the best of our knowledge, there are no studies concerning the actual toxicological relevance of this phenomenon.

Figure 2: Glutathione complexation with methylmercury.

The conjugation of glutathione with methylmercury is catalyzed by glutathione-S-transferases (event a), forming a methylmercury-glutathione conjugate that can be exported from the intra- to the extracellular environment via multidrug resistance-associated proteins (event b). Event (c) represents the transference of methylmercury from the methylmercury-glutathione complex to another thiol (i.e. sulfhydryl protein), leading to protein thiol oxidation.

GSH (reduced glutathione = γ-l-glutamyl-l-cysteinylglycine); GSTs (glutathione-S-transferases); ⁺HgMe (methylmercury); GS-HgMe (methylmercury-glutathione complex).

Based on the direct chemical interaction between MeHg and GSH, MeHg-induced GSH depletion is an expected event. Nevertheless, it is noteworthy that intracellular GSH levels are commonly in the millimolar (mM) range in several organs’tissues (liver, brain, kidney, etc.). Taking into account that GSH depletion has been observed in the brain of MeHg-exposed rodents whose brain mercury levels were in the low micromolar range [95,96], it is reasonable to suppose that MeHg-GSH interaction is not enough to completely explain MeHg-induced GSH oxidation.

3.2. MeHg decreases intracellular levels of GSH precursors

Peripheral cysteine delivered to the CNS is oxidized extracellularly to cystine [100], a disulfide formed by the oxidation of two cysteine molecules. Taking into account that cysteine is a limiting precursor for GSH synthesis, its intracellular levels are dependent on the availability of cystine [101]. With particular emphasis on the CNS, in vitro studies with cultured cells have shown that cultured astrocytes and hippocampal neurons can accomplish the uptake of both cysteine and cystine (for a review, see [102]). With respect to cystine, treatment of these cells (cultured astrocytes or hippocampal neurons) with MeHg causing decreased cystine uptake in astrocytes, but not in neurons [103]. With respect to cysteine, MeHg treatment also did not change cysteine uptake in neurons, although decreasing cysteine uptake in astrocytes [104]. Although MeHg-induced inhibition of cysteine and cystine uptake has not been observed in neurons, it is important to mention that MeHg could cause an indirect decrease of neuronal GSH synthesis as consequence of MeHg-induced changes in the astrocytic uptake of GSH; this is believed because astrocytes provide GSH precursors to neurons [105].

3.3. MeHg modulates enzymes involved in the GSH-GSSG redox cycle

A remarkable process related to the effects of MeHg on the GSH-GSSG redox cycle is its effect on GPx. Most of the GPx isoforms are selenoproteins, which present crucial selenocysteine residues (in the form of selenol group or selenolate: -SeH or -Se⁻) at their catalytic sites [75]. On the other hand, it is well known that mercurials, including MeHg, present a great affinity for selenols (greater than that for thiols) [106]. In fact, MeHg is a soft electrophile (or soft acid) that preferentially interacts with nucleophilic groups (mainly thiols and selenols) from proteins and low-molecular-weight molecules. On the other hand, these thiols and selenols (present in several molecules of the GSH antioxidant system: GSH, GPx, Grx) are soft nucleophilic groups (soft bases), which have high affinities for soft acids, such as MeHg [107]. Of note, selenium is a softer nucleophile compared with sulfur thiols and, therefore, selenols are more prone to be target by MeHg compared to thiols [108]. Accordingly, both in vitro and in vivo data indicate that the activities of different GPx isoforms, such as GPx1, GPx 3 and GPx 4, are decreased after MeHg exposure [24, 29,109–112]. With respect to mechanisms, in an in vitro study with cultured neurons, Farina and collaborators [24] reported that MeHg is a direct inhibitor of GPx1 likely due to the direct interaction of MeHg with selenocysteine, located at the enzymes active site. On the other hand, it seems that post-transcriptional and pre-translational phenomena related to mercury-selenium interaction are also linked to the decreased activity of GPx and other selenoproteins [113,114]. In this regard, it seems that mercurials can cause a depletion of selenium necessary for de novo synthesis of selenoproteins [115]. In fact, the biosynthesis of protein-incorporated selenocysteine occurs on its tRNA, and the pathway begins with the attachment of the amino acid serine to tRNA, which is further converted to selenocysteine by the addition of selenide (HSe⁻) [116]. Of note, MeHg is known to interact with HSe⁻ [117,118], decreasing its availability to be incorporated into selenoproteins [113].

With respects to GR, in vivo data with rodents indicate that the effects of MeHg toward GR are dependent on different variables, such as tissue and developmental life stage. In a study where adult male C57/BL6 mice were orally administered 5 mg/kg/day MeHg for 28 days, Kirkpatrick and collaborators [119] observed that GR activity decreased in liver and increased in brain tissue of MeHg-treated animals. An increase in brain GR activity was also observed in three independent studies [110, 120, 121], where adult Swiss mice were orally exposed to MeHg during 2-3 weeks. In line with these studies, it has been proposed that the increase in GR may represent a compensatory response to preserve GSH homeostasis [121]. In sharp contrast to the increased GR activity observed in the brain of MeHg exposed adult mice, studies with developmental (in utero or early post natal) exposures to MeHg have shown decreased GR activity in the CNS of mice pups [96, 122].

Based on the effects of MeHg on the enzymes involved in GSH-GSSG redox cycle (especially GPx), one might expect increased levels of peroxide and lipid peroxidation after MeHg exposures. In line with this, increased peroxide levels [123,124] and enhanced lipid peroxidation [120,125] have been vastly reported in the literature as a consequence of MeHg exposures. From a mechanistic point of view, an in vitro study with cultured mouse cerebellar neurons showed that MeHg causes significant inhibition of GPx activity, leading to a decreased ability to deal with exogenously peroxides, increased lipid peroxidation, and cell death [24]. On the other hand, evidence shows that MeHg has the capacity to increase the generation of hydrogen peroxide (or its precursor superoxide anion) in mitochondria [123,124]. Based on these evidences, it seems that the increased levels of hydrogen peroxide observed in MeHg exposed animals [126] represent a consequence of both increased generation and decreased detoxification.

The effect of MeHg on the GSH-GSSG redox cycle seems to be also important in MeHg-induced glutamatergic dyshomeostasis (for a review, see [23]), which is a significant phenomenon related to its neurotoxicity. In an in vitro study with cultured astrocytes, Allen and collaborators [25] showed that MeHg is able to inhibit astrocytic glutamate transporters, leading to increased glutamate concentrations in the extracellular fluid, and that this event was prevented by adding catalase. These results indicate that hydrogen peroxide, generated after MeHg exposure, caused the observed decrease in glutamate uptake in astrocytes. These in vitro results [25] are in line with in vivo evidences showing MeHg exposure caused changes in glutamate uptake by rodent cerebral cortex slices [127,128] and increased the levels of extracellular glutamate in the cortices of rats [129]. The most important events related to the effects of MeHg in the enzymes involved in GSH-GSSG redox cycle (with focus in the CNS), as well as related consequences, are depicted in Figure 3.

Figure 3: Methylmercury inhibits glutathione peroxidase and leads to increased levels of hydrogen peroxide, glutamate dyshomeostasis and lipoperoxidation.

Event (a) represents the glutathione peroxidase-catalyzed reduction of hydrogen peroxide to water with the simultaneous oxidation of glutathione to its disulfide form. Event (b) represents the astrocyte glutamate uptake, which is crucial to remove glutamate from the synaptic cleft, preventing excitotoxicity. As consequence of methylmercury-induced inhibition of glutathione peroxidase (event c), increased levels of hydrogen peroxide may lead to decreased glial glutamate uptake (event d) and increased lipoperoxidation (event h). As result of decreased astrocyte glutamate uptake, the increased levels of extracellular glutamate may lead to hyperactivation of its receptors (i.e. N-methyl-D-aspartate receptor) and increase in calcium influx (event e), which may culminate in excitotoxicity (event f) and mitochondrial dyshomeostasis (event g).

GSH (reduced glutathione = γ-l-glutamyl-l-cysteinylglycine); GSSG (oxidized glutathione); Glu (glutamate); GPx (glutathione peroxidase); H₂O₂ (hydrogen peroxide); NMDA R (N-methyl-D-aspartate receptor); mGlu R (metabotropic glutamate receptors).

3.4. MeHg and GSTs

Studies concerning the effects of MeHg in GSTs are scarce [110]; however, there is a significant amount of data (including in humans) reporting that GSTs affect MeHg elimination by catalyzing MeHg-GSH conjugation (Figure 2, event a). In experiments with rats, the treatment with pharmacological inhibitors of GSTs (benziodarone or azathioprine) significantly decreased the biliary transport of MeHg [130]. In a search for MeHg tolerance genes in Drosophila, Mahapatra and collaborators [131] identified three GST genes (GSTD3, GSTE3, and GSTE7) with higher expression in strains of flies that were more resistant to MeHg. In another study with Drosophila, Vorojeikina et al. [73] investigated a possible functional role for GSTs in modulating MeHg toxicity during development. The authors observed that RNAi knockdown of endogenous GSTD1, GSTE1, or GSTS1 increased susceptibility to MeHg in developing pupa, leading to decreased rate of adult eclosion. On the other hand, the exogenous expression of human GSTP1 in developing flies resulted in increased MeHg resistance. In addition, the authors observed a trend whereby Hg body burden was inversely related to the levels of GST activity [73].

The potential association between polymorphic variants of GSTs and MeHg levels in humans has also been reported. Concerning the GSTs from classes Mu (GSTM) and Theta (GSTT), whose deletions seem to impair catalytic activity leading to greater sensitivity to toxic compounds [132, 133], Barcelos and collaborators [134] investigated the genotype frequency of the GSTM1 and GSTT1 polymorphisms and examine the influence of the GSTM1*0 and GSTT1*0 polymorphisms (homozygous deletions] on hair and blood levels of Hg in a group of riparian individuals exposed to MeHg in the Amazon region of Brazil. The authors observed that GSTT1 genotype carriers presented lower levels of blood and hair Hg when compared to other genotypes carriers. Moreover, GSTM1*0/GSTT1*0 individuals presented higher Hg levels in blood and hair than subjects presenting any other genotypes.

Concerning the GSTs from class Pi (GSTP], two common polymorphisms in the coding region of the human GSTP1, Ile105Val [GSTP1₁₀₅] and Ala114Val [GSTP1₁₁₄], lead to lower catalytic activity compared to the wild-type GSTP1_wt. [135, 136] In studies with humans, some evidence has shown that carriers of the GSTP1₁₁₄ allele are more prone to present increased Hg levels in blood or hair [133, 137]. On the other hand, other studies reported that GSTP1₁₀₅ and GSTP1₁₁₄ carriers showed association with lower Hg retention [138, 139]. Although there are some contradictory results in the literature (mainly related to human studies), findings from experimental and population studies indicate a potential role for GSTs in moderating Hg body burden in MeHg-exposed subjects. The most important role of GSTs in MeHg metabolism is the catalysis of MeHg and GSH conjugation (Figure 2).

3.5. MeHg and glutaredoxins:

Studies on the effects of MeHg on glutaredoxins are scarce. In an in vitro study with CCF-STTG1 human astrocytoma cells, Robitaille and coworkers [140] observed that exposure to a low MeHg concentration (1 μM), which did not significantly change GSH levels, increased GSSG levels by approximately 12-fold, leading to reduced GSH/GSSG ratio. Moreover, the authors observed that the enzyme glutaredoxin-1, which is involved in reversing protein S-glutathionylation, was inhibited by approximately 50% after MeHg exposure. Consistent with this effect, the authors also observed that MeHg treatment increased the S-glutathionylation of high molecular weight proteins. This study [140] seems to be the first to show that treatment with low MeHg concentrations directly interferes with glutaredoxin-1 activity, thus prolonging protein S-glutathionylation. In another in vitro study with cultured cells, Branco and coworkers [141] observed that MeHg exposure (1 μM) decreased the activity of Grx activity (< 20%; p<0.05) in neuroblastoma cells (SH-SY5Y), although no change in the expression of either Grx1 or Grx2 was observed. In this same study [141] , experiments with primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) showed that such protein is important in protecting thioredoxin 2 from oxidation caused by another organic mercurial (ethylmercury). In this regard, a potential link between the effects of mercurials on the GSH system along with its effects on the thioredoxin system may have synergistic/additive toxicological relevance. To the best of our knowledge, there are no in vivo studies on the effects of MeHg on Grxs.

3.6. MeHg and Nrf2

The oxidation of critical cysteine residues from Keap-1 (Cys151, Cys273, Cys288 and Cys297) (see section 2.1) has been reported as an important event that activates the disruption of the Keap-1/Nrf2 association, favoring Nrf2 nuclear translocation [55]. In line with the electrophilic properties of MeHg, in vitro studies have reported the direct interaction of MeHg with the cysteine residues in Keap-1 structure [48, 142]. Accordingly, in vitro [143, 144] and in vivo [145] studies have shown that MeHg increases in the Nrf2 nuclear translocation. In accordance, MeHg has been reported to increase the expression of Nrf2-related genes, such as ho-1, cglc, nqo1 and xCT [143, 145]. In general, the aforementioned studies indicate that MeHg is able to interact with (and oxidize) critical cysteine residues of Keap-1, stimulating Nrf2 nuclear translocation and, consequently, favoring the expression of Nrf2-related genes. In addition to the oxidation of critical cysteine residues of Keap-1, phosphorylation events have also been reported as modulators of MeHg-induced Nrf2 nuclear translocation. In this regard, MeHg seems to stimulate Nrf2 activity by downregulating the Src family kinase Fyn [146] and by modulating phosphatidylinositol 3 (PI3) kinase [144]. For mechanistic details concerning phosphorylation events and signaling pathways modulating MeHg-induced Nrf2 nuclear translocation, see Antunes dos Santos et al. [147] and Unoki et al. [148].

The relationship between MeHg and Nrf2 is not only related to the stimulatory effects of MeHg toward Nrf2 nuclear translocation, but also to the protective effects of Nrf2 activation against MeHg toxicity. In this context, several compounds that are known to stimulate Nrf2 nuclear translocation, increasing the expression of Nrf2-related genes (i.e., sulforaphane [145], isothiocyanates 6-methylsulfinylhexyl isothiocyanate [149], hydroxytyrosol [150]) have been reported to ameliorate some toxic effects induced by MeHg. In this scenario, it seems that the activation of Nrf2 induced by MeHg is linked to increased protection against this compound [48].

4. Concluding remarks and perspectives

As previously discussed, MeHg is an electrophilic compound that interacts with thiol and selenol groups of several biomolecules. Even though MeHg can form a relatively stable chemical interaction with thiols and selenols, it can be exchanged between different nucleophilic groups [97], allowing for its transfer between different molecules. In this regard, there seem to be preferential targets for MeHg, which present nucleophilic groups with high affinity for its electrophilic core (-Hg⁺). Because GSH and some GSH-related enzymes (i.e. GPx, Grxs) represent some of these targets, MeHg affects multiple branches of GSH homeostasis, which has been extensively reported to have toxicological relevance [21].

Because of the low-dose and chronic profile of most cases of human exposure to MeHg, clear signs of toxicity are commonly detected only when important neurotoxic damage is achieved, at which point it is commonly irreversible. In this regard, it is important to identify and validate biomarkers that are predictive of early adverse effects prior to adverse health outcomes becoming irreversible [151]. Based on the relative high affinity of MeHg for GSH, one might propose this tripeptide as potential biomarker of MeHg exposures. However, many other compounds (i.e., paraquat, arsenic, benzo(a)pyrene, and acetaminophen) may also decrease GSH levels [151]. Consequently, in a multi-contaminant or therapeutic context GSH does not permit distinction of MeHg-related toxicity from that produced by other compounds. With respect to the use of GPx as a potential biomarker for MeHg exposure/poisoning, Usuki and Fujimura [111] have pointed out the plasma selenium-dependent glutathione peroxidase (GPx3) as a potential biomarker of ongoing MeHg cytotoxicity. Nevertheless, it is important to note that the activity of most of the GPx isoforms depends on the selenium status, which may be significantly affected by nutritional and regional characteristics. The measurement of MeHg-targeted GPxs (i.e., plasma GPx3) could represent a perspective related to the topic of “potential biomarkers” for MeHg exposure. However, studies are necessary to elucidate temporal aspects concerning the fate of MeHg after interaction with GPx3

Although the use of pharmacological strategies to recover patients from neuropathological conditions resulting from exposure to MeHg is impractical/ineffective, some lines of evidence point to the benefit of using antidote strategies to eliminate the toxicant from the body. In this context sulfhydryl-containing molecules display significant effects in increasing MeHg excretion from the body, at least in cases of acute exposure [152]. Even though GSH presents relative high affinity for MeHg, its use as antidote is not useful because GSH does not effectively enter the cells. On the other hand, N-acetylcysteine (a GSH precursor), as well as D-penicillamine and 2,3-dimercaptopropane sulfonate (DMPS), enhance MeHg excretion from the body [93,152]. Considering that MeHg has a higher affinity for selenols compared to thiols, the use of selenocompounds to remove MeHg from the body represents a perspective. In line with this, an in vivo study showed that diphenyl diselenide, an organic selenium compound, decreased Hg levels in several organs of MeHg-treated mice [153]. To the best of our knowledge, studies concerning the use of selenocompounds to increase MeHg excretion in humans have never been performed and represent a perspective.

An interesting aspect on the interaction of MeHg with GSH (or with GSH-related proteins, such as GPx) is that from a toxicological point of view, it represents a double-edged sword. In fact, on one hand the chemical interaction of MeHg with either GSH or GSH-related enzymes triggers toxicity [109, 121], while on the other hand such interaction is important in protecting against toxicity, favoring MeHg excretion [93]. In fact, the chemical interaction of MeHg with the GSH’s thiol group and/or with GPx’s selenol group decreases the cellular capacity to detoxify oxidants, favoring toxicity [24, 91]. On the other hand, MeHg excretion is significantly favored by its interaction with GSH [154]. This reciprocal relationship, which modulates toxic and protective pathways, highlights the intriguing interplay between MeHg and the GSH antioxidant system.

Highlights.

Methylmercury (MeHg) is toxic to several organs, but the central nervous system represents a preferential target especially during development (prenatal and early postnatal periods).

MeHg interacts with (and oxidize) nucleophilic groups present in proteins or low-molecular weight molecules and such interactions modify the redox state of these groups, leading to oxidative stress, impaired function and neurotoxicity.

Glutathione (GSH) and thiol- or selenol-containing enzymes belonging to the GSH antioxidant system represent key molecular targets involved in MeHg-neurotoxicity.

The chemical interaction of MeHg with the GSH’s thiol group and/or with GPx’s selenol group decreases the cellular capacity to detoxify oxidants, favoring toxicity.

MeHg excretion is significantly favored by its interaction with GSH