The Role of mTOR, Autophagy, Apoptosis, and Oxidative Stress During Toxic Metal Injury

C H A P T E R

5 The Role of mTOR, Autophagy, Apoptosis, and Oxidative Stress During Toxic Metal Injury Sarmishtha Chatterjee, Chayan Munshi and Shelley Bhattacharya Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati (A Central University), Santiniketan, India

5.1 INTRODUCTION

mTORC1, and mTORC2 binds to rictor. Signaling through mTORC1 is much better understood than signaling through mTORC2. mTORC1 is an important node in cellular regulation impacting on cell growth that is linked to aging [15,16]. Structurally, mTOR contains 2549 amino acids and the region of first 1200 N-terminal amino acids contains up to 20 tandem repeated HEAT (a protein
protein interaction structure of two tandem antiparallel α-helices found in Huntingtin Elongation factor 3, PR65/A subunit of protein phosphatase 2A, and TOR) motifs [17]. HEAT repeat region is followed by a FAT [FRAP, ATM, and TRRAP (PIKK family members)] domain and FKPB12
rapamycin binding domain, which serves as a docking site for the rapamycin
FKBP12 complex. Downstream lies a catalytic kinase domain and a FATC (FAT carboxyterminal) domain, located at the C-terminus of the protein [18]. The interactions between FAT and FATC might contribute to the catalytic kinase activity of mTOR [19]. The domain structure of mTOR is sketched in Figure 5.1.

Target of rapamycin (TOR) discovered in 1991, is a highly conserved protein kinase, essential for both fundamental and clinical biology [1]. The story of TORsignaling begins with a remarkable drug, rapamycin [2]. Rapamycin is a lipophlic macrolide as well as a natural secondary metabolite, produced by Streptomyces hygroscopicus. S. hygroscopicus is a bacterium, isolated from soil in Easter Island, called Rapa-Nui in 1965, thus the name rapamycin [1,3] was coined. Intracellular rapamycin receptor in all eukaryotes is a small ubiquitous protein, called FK506 binding protein 12 kDa (FKBP12) [4,5]. Rapamycin
FKBP12 complex interacts with evolutionarily conserved TOR proteins to potently inhibit downstream effectors. A single mammalian TOR (mTOR) protein was cloned from several species and alternatively termed as mTOR, FKBP12 and rapamycin associated protein (FRAP), rapamycin and FKBP12 target, sirolimus effector protein, or rapamycin target [6
11]. mTOR is 289 kDa, sharing B45% identity with the Streptomyces cerevisiae TOR proteins and B56% identity with Drosophila melanogaster TOR as well as the human, rat, and mouse mTOR proteins share .95% identity at the amino acid level [9,12]. The TOR proteins function as Ser/Thr protein kinases assigned to a protein family termed the phosphatidylinositol kinase-related kinases (PIKKs) [13,14]. mTOR is a multidomain protein kinase that interacts with other proteins to form two main types of complex, mTOR complexes 1 and 2 (mTORC1 and mTORC2) [15]. mTORC1 associates with raptor, which binds to proteins that are direct substrates for

Molecules to Medicine with mTOR DOI: http://dx.doi.org/10.1016/B978-0-12-802733-2.00013-X

5.2 CELLULAR FUNCTIONS OF mTOR mTOR is involved in many different cell functions to regulate cellular protein anabolism and catabolism [20,21]. If the cell has enough available amino acids, mTOR is free to signal to other molecules in the cell to build new proteins. On the other hand, if the cell is running low on nutrients, existing proteins and other cell components break down to free the building blocks which can be reused. The process by which the

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5. THE ROLE OF mTOR, AUTOPHAGY, APOPTOSIS, AND OXIDATIVE STRESS DURING TOXIC METAL INJURY

cell breaks down its own components is called autophagy [22], programmed cell death type II, which basically means “eating of the self” [23
26]. The degraded part of the cell is engulfed by a double membrane called phagophore, to separate from the rest of the cell, resulting in a membrane-enclosed bubble of cytosol, called autophagosome. Autophagosome eventually fuses with lysosomes, forming autophagolysosomes as the garbage disposal of the cell that can break down all sorts of cellular components. Key regulators of autophagy include the class I phosphoinositide-3kinase (PI3K) [27] and adenosine-mono-phosphate activated protein kinase (AMPK) [28] in response to TOR kinase (Figure 5.2).

FIGURE 5.1 The domain structure of mTOR.

Activated PI3K signals TOR kinase, especially mTORC1 via protein kinase B (Akt kinase, belonging to the AGC protein kinase family) and modulates autophagy [22,29]. AMPK regulates autophagy through the control of mTORC1. AMPK indirectly inhibited TORC1 by phosphorylation of tuberous sclerosis complex-2 (TSC2) via deactivation of the Rheb GTPase (Ras homology enriched in brain-GTP binding protein) [30
32]. AMPK activation also induces phosphorylation of p53 on Ser15 domain. Phosphorylated p53 inhibits mTORC1 activity and regulates its downstream targets including autophagy [33
35]. In addition, mTOR has profound effects on the control of apoptosis [36]. Apoptosis, programmed cell death type I, plays an important role in various physiological and pathophysiological circumstances [37]. The intrinsic pathway of apoptosis deals with mitochondrial pathway, is initiated by a large variety of upstream stimuli and tightly modulated by various factors including, pro- and antiapoptotic proteins of the Bcl-2 family as well as PI3K/Akt/mTOR pathway [38
40]. The Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial permeability. The proapoptotic Bcl-2 proteins [Bcl-2 associated X-protein (BAX) and proapoptotic BH3 only Bcl-2 family protein (BIM)] may reside in the cytosol but translocate to

FIGURE 5.2 Role of mTOR in autophagy.

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5.3 FREE RADICALS IN CELLS

FIGURE 5.3 Role of mTOR in apoptosis.

mitochondria following death signaling and promote the release of cytochrome c (Cyt c) [40]. The PI3K signaling network diversifies into many distinct downstream branches, one of which leads to the phosphorylation of mTORC1 [41
45] on Thr 2446 and Ser 2448 sites by Akt kinase via TSC2 phosphorylation. Akt acts as an antiapoptotic factor that directly or indirectly antagonizes cell death signal transduction via the mitochondrial pathway [46]. Akt directly interferes with the phosphorylation of apoptosis-regulatory proteins which in turn results in a shift to pro- and antiapoptotic stimuli [47
49]. The multidomain Bcl-2 protein BAX and BIM are phosphorylated at serine residues Ser 184 and Ser87, respectively, in an Aktdependent manner [49,50]. Akt-mediated direct and indirect (by mTORC1) phosphorylation of some antiapoptotic factors, such as myeloid leukemia cell sequence 1 (Mcl-1) located in mitochondrial matrix and vice versa, sometimes diminishes the antiapoptotic properties by decreasing their protein stability. Thus degradation of these proteins via the proteasomal machinery results in a reduction of Mcl-1 protein expression [48,51] which signals the release of Cyt c from mitochondria and induces the intrinsic apoptotic pathway. On the other hand, mTORC2 elicits its activity as a potent antiapoptotic signal through Mcl-1 [52] and Akt phosphorylation [45,53]. mTORC2 phosphorylates Akt on Ser 473 and Thr 308 region by protein-dependent kinase 1 (PDK1), however mTORC2 recognizes Ser 473 region in the hydrophobic motif of Akt as a kinase [53,54] and thereby converts inactive Akt to active Akt. Phosphorylated Akt/active Akt migrates to cytosol,

mitochondria, and nucleus. Nuclear Akt then fulfills the antiapoptotic role [55] through phosphorylation of transcription factors such as cAMP response elementbinding (CREB) protein [40,56,57]. Phosphorylated CREB binds to DNA as a dimer via leucine zipper motif and recognizes cAMP-response elements (CREs). CRE then stimulates the transcription of genes and results in the expression of the antiapoptotic factor, Bcl-2 (Bcl2-xL) [58]. The regulation of apoptosis by PI3K/Akt/mTOR pathway is schematically depicted in Figure 5.3.

5.3 FREE RADICALS IN CELLS Living cells are always subjected to the hazardous effects of exogenously or endogenously produced highly reactive oxidizing molecules and easily acquire electrons from oxidized molecules with which they remain in contact, such as all cellular biomolecules, generating chain reactions and ultimately leading to cell structure damage [59]. Products of oxidative metabolism that provoke oxidative injury are collectively called reactive oxygen species (ROS), which includes superoxide, H2O2, and the hydroxyl radical. They are formed either by the loss of a single electron from a nonradical or by the gain of a single electron by a nonradical. Lipid peroxidation is one of the wellstudied consequences of ROS. Lipid peroxidation initiators, such as ROS, contribute to the signal transduction cascade that controls cell death and proliferation [60]. The free radicals can trigger cellular damage by covalent binding to macromolecules and enhance

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lipid peroxidation that depletes the stress of antioxidant enzymes. Inhibition of antioxidant enzymes leads to oxidative stress [61,62]. ROS has a major biological impact due to its endogenous production and high concentration in the cells. It is well known that the principal source of ROS in the cell is the mitochondrial respiratory chain [59,63]. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhances lipid peroxidation, and alters calcium and sulfhydryl homeostasis [64]. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal, and other exocyclic DNA adducts (etheno and/or propano adducts). While iron (Fe), copper (Cu), chromium (Cr), and cobalt (Co) undergo redox-cycling reactions, for mercury (Hg), cadmium (Cd), and nickel (Ni), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins [64,65]. Metal toxicity therefore generates oxidative stress in cell.

` -VIS mTOR 5.4 STRESS VIS-A The TOR pathway is an evolutionarily conserved signaling module regulating cell growth in response to a variety of internal and external stimuli, generating oxidative stress [66,67]. mTOR acts in two categories in the surveillance of stress conditions, illustrated in Figure 5.4. The oxidative stress-input to mTOR

generally lies upstream of the TSC complex. ROS induces transcriptional alteration of cellular proteins and impinges the mTOR network signaling [66]. ROS in turn inhibits mTORC1 by direct phosphorylation of TSC2 and indirect activation of AMPK. AMPK results ROS generation, phosphorylates, and thereby activates TSC2, resulting in mTORC1 inhibition [31]. On the contrary, oxidative stress can persuade mTORC1 to play a distinct role in the last stage of autophagy [68,69]. Generally, mTORC1 forms a complex with autophagy-related proteins (ATG) named ATG1, ATG 13, and ATG101, which in turn phosphorylates ATG1 and ATG13 to inhibit the ATG activity in autophagy [28,70]. Induction of stress can reactivate mTORC1 and thereby breaks the mTORC1
ATG protein complex to free ATG1 [69]. ATG1 then leads to the formation of protolysosomal extension by lysosomal-associated membrane protein 1 and microtubule-associated protein 1A/1B light chain 3 (LC3) from autophagolysosome and ultimately mature into functional lysosome by autophagic lysosome reformation [68,69,71]. Toxicants can trigger cellular pathways classified broadly as death and survival signals. Each organ has its own critical threshold towards a toxicant, regulated by an intricate signaling system. Depending on the concentration and duration of exposure to toxicants, cells can utilize a coordinated, preprogrammed signaling pathway to maintain the normal homeostasis of the organ. Considering the complex and interactive mechanisms involved in maintenance of homeostasis of the organism, the present review is further aimed to FIGURE 5.4 mTOR.

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Oxidative stress and

5.6 CADMIUM (CD) TOXICITY

monitor the role of mTOR during cellular injury imposed by toxic metals. Generally, oxidative stress is considered as the first response to toxic metals and a volley of interactions follows to maintain the integrity of the cellular and molecular homeostasis through several coordinated signaling pathways. Toxic metals are usually those which are highly reactive to the lipid membranes of the cells, resulting in formation of injurious free oxygen or hydroxyl radicals. An attempt has been made to weigh the deleteriousness of metals of environmental concern affecting biological systems and the interrelationship of mTOR with different types of cell death.

5.5 ARSENIC (AS) TOXICITY The main cause of toxic effects of metals is generation of ROS. It has been demonstrated that As is a potential inducer of oxidative stress as evidenced by DNA damage, by superoxide and hydrogen peroxide [72], and by disruption of mitosis while promoting apoptosis [73,74]. It has been amply demonstrated that inorganic As is more toxic than organo-As compounds [75]. Moreover, arsenite induces apoptosis much more strongly than arsenate [76,77] and there is a definite role of free radicals in As toxicity [78]. Various studies on the role of As in cellular apoptosis demonstrate 44.5% apoptosis at a concentration of 200 μM arsenite of the JB6C141 cell types [79]; 10 and 20 μM As2O3 initiated apoptosis in a neural cell line and 40 μM caused death in these cells [80]. Paradoxically, As has both carcinogenic [81,82] and effective chemotherapeutic activity [83
85] in acute promyelocytic leukemia (APL), inducing apoptosis within a tumor cell population where a high cumulative dose (340
430 mg) of As2O3 was found to cause a complete remission of APL [86]. Thus, normal organs and tissues are liable to As toxicity evidenced by triggering of signaling pathways, such as caspasedependent or -independent, mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), and p38 pathways. Arsenic-induced apoptosis at pharmacological concentrations was mediated by caspase-dependent pathways through mitochondrial checkpoints at 10 μM As2O3 where the activity of ERK and JNK is dependent on caspase activity, while p38 and MAPK were found to be operative in inducing apoptosis at 40 μM of As2O3 [87]. As-induced apoptosis of chronic lymphocytic leukemia cells involves inactivation of the kinase AKT and a blockade of the transcriptional factor NF-κB, as well as up-regulation of PTEN and downregulation of X-linked inhibitor of apoptosis protein [88]. As can paradoxically increase mTOR activation

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and engagement of downstream mTOR effectors in BCR-ABL (tyrosine kinase inhibitor) expressing cells [89] or acute myeloid leukemia cells [90]. The combinations of As with mTORC1 inhibitor (mTORC1i) of rapamycin result in increased apoptosis and enhanced suppressive effects on primary leukemic progenitors [91]. Moreover, the combination of As and mTORC1i may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways in breast cancer [92]. Since carcinogenesis and apoptosis are both tightly linked to the PI3K/Akt/mTOR pathway, it could be expected that As31 would differentially target the pathway to accomplish these opposite effects on cell fate [93]. As2O3 has been reported to induce autophagic cell death without activation of caspase-dependent apoptotic cell death [94
96]. Arsenic was reported to induce autophagy at a concentration of 2 μM in malignant glioma cell lines [94] and at 6 μM in lymphoblastoid cell lines [97]. Autophagy plays complex roles in Asinduced death of human promyelocytic leukemia 60 cells; it inhibits As-induced apoptosis in the initiation stage, but amplifies As-mediated apoptotic program on persistent activation of autophagy [98]. Furthermore, carcinogenicity of As can fabricate overproduction of interleukin 6, which sufficiently blocks autophagy by supporting Beclin-1/Mcl-1/mTOR interaction [99].

5.6 CADMIUM (CD) TOXICITY Cadmium (Cd), the toxic transitional metal, is mainly released from smelting and refining of metals and cigarette smoking, resulting in the pollution of water, air, and soil [65,100]. Cd compounds are used as stabilizers, color pigments, rechargeable batteries, alloys, and can be found in some fertilizers. Cd production, consumption, and emissions to the environment increased worldwide dramatically during the twentieth century [101]. Cd can be absorbed and accumulated in plants and animals and thereby can be accumulated in the human body either through direct exposure to Cd-contaminated environment or by the food chain. Such accumulation contributes to carcinogenesis, immunodepression, and neurodegeneration [100,102,103]. A very long biological half-life of Cd may be responsible for the accumulation in many human organs, including kidney [104], liver [105], lung [106], testis [107], bone [108], and blood system [109]. Cd has a high blood
brain barrier permeability; therefore chronic Cd exposure affects the nervous system, including learning disabilities and hyperactivity in children [110,111], olfactory dysfunction, neurobehavioral defects in attention, psychomotor

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speed, and memory in industrial workers [112] and also Parkinson’s disease by acute Cd poisoining [113]. Cd toxicity is amplified as a consequence of the long biological half-life of the metal and has been associated with blockage of oxidative phosphorylation, glutathione depletion, inhibition of antioxidant enzymatic activity, production of oxidative stress, DNA damage, reduction of protein synthesis, and cell death [65,114]. Cd-induced toxicity is closely associated with the production of ROS [115
117]. Cd accelerates the generation of ROS by depleting cellular glutathione and antioxidant enzymes, such as superoxide dismutase and catalase [115,118] and by inhibiting the electron transfer chain in the mitochondria [119]. It is surmised that the effect of Cd toxicity depends on its concentration and the duration of exposure inducing both autophagic- and apoptotic-related pathways via ROS generation [65,120,121]. However, the mechanisms underlying Cd-induced autophagy are not yet completely understood. Cd (0
10 μM) has been found to induce autophagy through ROS-dependent activation of the serine/threonine kinase 11/liver kinase B1—AMPK—mTOR signaling in skin epidermal cells in a concentration- and time-dependent manner [122]. Cd could also provoke oxidative stress generating ROS inside JB6 cells, which in turn signals energy-sensing LKB1-AMP-activated protein kinase pathway after 12 h of Cd treatment (10 μM) and the effect is further augmented at 24 h after the treatment. AMPK, a heterotrimeric protein complex, is downstream of LKB1 in a signaling pathway that regulates energy homeostasis [123,124]. LKB/AMPK signaling can act as the upstream of mTOR signaling [125,126]. Cd-induced intracellular level of ROS signals LKB1, which in turn phosphorylates AMPK and thus inhibits mTORC1 triggering autophagy in JB6 cells in a significant manner [123,124]. It is commonly accepted that Cd induces apoptotic cell death in cellular systems [127,128]. However, Cd induces apoptosis by either caspase-dependent [129,130] or caspase-independent mechanisms [131,132] depending on the target cell type. Cd-induced apoptotic cell death in JB6 cells was found to be dependent on Ca21and H2O2-mediated JNK and p53 signaling [133]. Interestingly, autophagic cell death was also induced by Cd (0
10 μM) by the ROS-dependent LKB1-AMPK [122]. Moreover, 10 μM of Cd not only causes apoptosis in skin epidermal cells (JB6) but also leads to necrotic cell death [133] in a time-dependent manner from 1 to 24 h. Generation of ROS due to Cd toxicity elevates the intracellular calcium (Ca21) level. The increase of free calcium ions is believed to be one of the main mechanisms involved in Cd-induced apoptosis by stimulating the generation of H2O2 [133]. Ca21 regulates JNK signaling and also effects H2O2 generation. In turn H2O2 generated

by Cd regulates both activator protein-1 (AP-1) and p53 pathways in JB6 cells. Collectively, Cd promotes apoptosis mainly through Ca21-JNK-growth arrest and DNA-damaged inducible protein (GADD45α) as well as a caspase-independent pathway, H2O2-AP-1-p53-apoptosis-inducing-actor (AIF) signaling cascades [133]. Ca21 also leads to the activation of MAPKs in Cdexposed mesangial cells [120] and in tributyltin-treated human T-cell lines [134]. It has been reported [100,135] that Cd at 0
120 mmol/L and 10 and/or 20 μM, respectively, induced neuronal apoptosis by the activation of MAPK and mTOR as well as JNK and PTEN-Akt/ mTOR network. Cd toxicity activates ROS production mediated by NADPH oxidase, which in turn activates MAPK [136
138]. Ca21-dependent protein kinase and PI3K also stimulate MAPK during Cd toxicity [138]. Phospho-p38-MAPK signals GADD45α and induces apoptosis by caspase-3 activation [136,139,140]. JNK and PTEN-Akt/mTOR network diminishes antiapoptotic properties by decreasing their protein stability, which eventually releases Cyt c from mitochondria and induces caspase-dependent apoptosis [51,135]. The effects of Cd toxicity are schematically summarized in Figure 5.5.

5.7 LEAD (PB) TOXICITY Lead (Pb), a well-known heavy metal, can cause pathophysiological changes in several organ systems including the central nervous system (CNS), cardiovascular, hematopoietic, reproductive, gastrointestinal, and renal systems [141,142]. Pb had modest early uses in ancient medicines and cosmetics, however, in the modern era it has had industrial uses in building materials, paints, and gasoline [143]. Pb has become ubiquitous in the environment. Pb exposure mainly occurs through the respiratory and gastrointestinal systems and absorbed Pb is stored in soft tissues. Inhaled Pb (B30
40%) enters the bloodstream [144]; once absorbed, 99% of Pb is retained in the blood for approximately 30
35 days and over the following 4
6 weeks it is dispersed and accumulated in other tissues [145]. Pb serves no useful purpose in the human body and its unfortunate presence in the body can lead to toxic effects, regardless of the exposure pathway adopted [146]. The effects of Pb on Ca21 fluxes and Ca21-regulated events are the major mechanisms of lead neurotoxicity [142,147,148]. Pb can cause abnormal hyperphosphorylation of tubulin-associated unit (tau) protein and accumulation of α-synuclein, inducing hippocampal injury affecting the ability to learning and causing memory damage. Pathological hyperphosphorylation and aggregation of tau deposits as neurofibrillary tangles in the brains of those with Alzheimer’s disease and other related neurodegenerative disorders are called tauopathies [142,149]. α-Synuclein,

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5.8 MERCURY (HG) TOXICITY

FIGURE 5.5

expressed in the CNS neurons and localized around synaptic vesicles in presynaptic terminals can cause Parkinson’s disease or a related disorder, dementia with Lewy bodies through genetic alteration or point mutation [142,150]. The deleterious effects of Pb exposures can involve endoplasmic reticulum (ER) stress in cardiofibroblasts which consecutively generate ROS and a direct depletion of the antioxidant reserves [145,148]. Significant effects have been found on various fundamental cellular processes like protein folding and maturation, ionic transportation, enzyme regulation leading to cell stress [151], and finally cell death, such as autophagy and apoptosis [142,148]. ER has an essential role in multiple cellular processes, such as the folding of secretory membrane proteins, calcium homeostasis, and lipid biosynthesis. When ER trans-membrane sensors detect the accumulation of unfolded proteins, the unfolded protein response (UPR) is initiated to cope with the resulting ER stress [152]. Under stress conditions, a cell performs several adaptive alterations such as autophagy and apoptosis. Pb toxicity can induce robust UPR and ER stress which can mediate autophagy and/or apoptosis to protect the internal system of the cell [142,148]. The regulation of autophagy and to some extent apoptosis is mediated by mTORC1 [32,36]. mTORC1 activity is critical for de novo protein synthesis; the reduced mTORC1 activity may decrease the rate of synthesis of new proteins, which may partly attenuate ER stress on protein folding and processing reactions [148]. Recently it has been reported that Pb

Toxic signals of Cd.

toxicity inhibits the mTORC1 pathway [142,148]. Depending on the concentration and duration of Pb exposure, the internal cellular system then decides to undertake the cellular homeostatic pathway, autophagy/ apoptosis, mediated by the ATG proteins. Inhibition of mTORC1 easily shifts the cell towards the induction of autophagy via ATG1
ATG13
ATG101 complex and Beclin-1 (ATG6)
ATG14 complex [23,25]. On the other hand, Beclin-1 plays a significant role in the network of cellular homeostasis, focusing on the cross-regulation between autophagy and apoptosis [153,154]. Pb toxicity can induce apoptosis to promote the activation of caspase-3 via Beclin-1 [142]. It cleaves Beclin-1 from the Beclin-1
ATG14 complex, triggers proapoptotic proteins in mitochondria to release Cyt c which consequently induces apoptosis [153]. The effects of Pb toxicity are portrayed in Figure 5.6.

5.8 MERCURY (HG) TOXICITY Hg is a well-established environmental toxicant. Hg is known to cause several physiological and biochemical disturbances in mammals. Most of these biochemical reactions are exerted through formation of covalent bonds between sulfur and Hg. Three forms of Hg exist: elemental, inorganic, and organic. Each of them has its own profile of toxicity. Elemental Hg vapor is highly lipid-soluble allowing it to readily cross cellular membranes. It can also be oxidized to mercuric salts from monovalent Hg

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FIGURE 5.6 Toxic signals of Pb.

compounds. Thus, when ingested, Hg will be more rapidly absorbed and produce greater toxicity [155]. The distribution kinetics of radioactive Hg in different hepatocellular fractions revealed that Hg treatment increases nuclear and liposomal protein content significantly [156]. On the other hand, generation of an acutephase response was observed in rabbit treated with mercuric chloride (HgCl2) and remarkable changes in phospholipid profile and acetylcholinesterase activity, [157] while in rat Hg has been found to induce C reactive protein along with a metallothionein-like protein [158]. HgCl2 is also known to damage DNA in fibroblasts of rat and mouse embryos [159,160] and exposure to a nonlethal dose of Hg induces oxidative-stress-mediated apoptosis in rat liver [87,161]. Five micromolar HgCl2 was shown to damage cellular DNA by a nonapoptotic mechanism in the U-937 cell line [162]. Interestingly, recent research [35,163
165] revealed that 5 μM HgCl2 drives autophagy and apoptosis in different cell types of rat liver. Covalent-conjugation of autophagic proteins (ATG5-ATG12-LC3B) stimulates cell death in rat hepatocytes, modulated by Keap1-p62, ERK-p38, and DRAMp53 regulator proteins through inhibition of mTOR [35,164]. Concurrently, autophagic proteins ATG12 and LC3B exerted a significant role in triggering apoptosis in 5 μM HgCl2-treated rat oval cells [165].

pathways. Oxidative stress and PI3K, Akt, and mTOR pathways are intimately related to determine the cell fate [166]. The significant biological role of PI3K, Akt, and mTOR is involved in the modulation of apoptosis, autophagy, disorders of cellular metabolism, acute nervous system injury, and chronic neurodegeneration [20,21,166
168]. Moreover, MTOR activates CREB1 through suppression of autophagy, sensitizing subsequent apoptosis [169]. Different metals seem to prefer different signaling pathways and display diverse potential to trigger mTOR pathway cascades [49,135,148]. mTOR signaling pathways integrate both intracellular and extracellular signals and serves as a central regulator of metal toxicity in cellular adaptation. In conclusion, this review elucidates an important role for mTOR modulation of oxidative stress and cell death signaling due to toxic metal injury.

5.10 FUTURE PERSPECTIVES Metal-induced oxidative stress plays a significant role in the modulation of apoptosis and autophagy. In this aspect, the PI3K, Akt, and mTOR cascade offers new inroads to the development of novel therapies. Nowadays, with increasing use of nanoparticles in the cosmetics and food industries, attention needs to be focused on metal-nanoparticle-induced oxidative stress and PI3K/Akt/mTOR signaling pathway. It appears to be an important mechanism in manifesting pathways leading to several cardiovascular diseases. New research is focusing on mTOR signaling components in the maintenance of insulin signaling to prevent diabetes mellitus. However, mTOR signaling can also trigger cellular proliferative pathways that can promote aggressive tumor growth. On the other hand, prevention of PI3K and Akt activation can block medulloblastoma growth. Toxicology studies have unraveled that the PI3K/Akt/mTOR pathway is commonly targeted by toxins, especially metals, to produce several hazardous conditions. The PI3K/Akt/mTOR targeted therapy shows therapeutic efficacy, albeit with a variety of toxic side effects. A thorough understanding of the role of the mTOR pathway, with a systemic perspective and computational modeling appears to be the future center of interest.

5.9 CONCLUDING REMARKS The last decade has seen a rapid rise in interest in mTOR signaling cascades. Metal affronted oxidative stress determines the down-regulation and upregulation of mTOR signaling to maintain cellular homeostasis by different programmed cell death

Acknowledgments SC is grateful to the National Academy of Sciences, India (NASI), for the award of Research Associate and SB acknowledges NASI for the award of Senior Scientist Platinum Jubilee Fellowship. The authors are grateful to Shuvasree Sarkar, UGC-BSR SRF, for critically going through the manuscript.

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