Mitochondrial Oxidative Stress Is the General Reason for Apoptosis Induced by Different-Valence Heavy Metals in Cells and Mitochondria

This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of different-valence heavy metals (Ag+, Tl+, Hg2+, Cd2+, Pb2+, Al3+, Ga3+, In3+, As3+, Sb3+, Cr6+, and U6+). The problems of the relationship between the integration of these toxic metals into molecular mechanisms with the subsequent development of pathophysiological processes and the appearance of diseases caused by the accumulation of these metals in the body are also addressed in this review. Such apoptosis is characterized by a reduction in cell viability, the activation of caspase-3 and caspase-9, the expression of pro-apoptotic genes (Bax and Bcl-2), and the activation of protein kinases (ERK, JNK, p53, and p38) by mitogens. Moreover, the oxidative stress manifests as the mitochondrial permeability transition pore (MPTP) opening, mitochondrial swelling, an increase in the production of reactive oxygen species (ROS) and H2O2, lipid peroxidation, cytochrome c release, a decline in the inner mitochondrial membrane potential (ΔΨmito), a decrease in ATP synthesis, and reduced glutathione and oxygen consumption as well as cytoplasm and matrix calcium overload due to Ca2+ release from the endoplasmic reticulum (ER). The apoptosis and respiratory dysfunction induced by these metals are discussed regarding their interaction with cellular and mitochondrial thiol groups and Fe2+ metabolism disturbance. Similarities and differences in the toxic effects of Tl+ from those of other heavy metals under review are discussed. Similarities may be due to the increase in the cytoplasmic calcium concentration induced by Tl+ and these metals. One difference discussed is the failure to decrease Tl+ toxicity through metallothionein-dependent mechanisms. Another difference could be the decrease in reduced glutathione in the matrix due to the reversible oxidation of Tl+ to Tl3+ near the centers of ROS generation in the respiratory chain. The latter may explain why thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc.


Introduction
Early research on heavy metal toxicity. This section is dedicated to a summary of heavy metal toxicity studies that have been performed in previous years. This summary will allow the reader to trace the evolution of the efforts of various researchers on this issue and to understand why the emphasis has shifted from the research of isolated mitochondria to the study of various cellular systems in vitro and in vivo.
Ag(I). It was previously shown that intense rat liver mitochondria (RLM) swelling was induced after the reaction of Ag + with 12% rapidly reacting SH groups of the inner membrane [1]. Ag + inhibited Na + , K + -ATPase [2], and mitochondrial malate dehydrogenase from bovine brain [3]. Ag + increased K + efflux, coincident with net Ca 2+ uptake, and it stimulated the ouabain-sensitive oxygen consumption rate in experiments with rabbit renal cortical tubule suspensions [4,5]. Thiol reagents (n-ethylmaleimide (NEM), dithiothreitol (DTT), and reduced glutathione) reversed these Ag + effects. chondrial swelling additionally increased in Ca 2+ -loaded RLM, which was followed by the inhibition of CsA [88].
Silver nanoparticles (AgNPs) have found wide application in both biomedical and consumer products [89]. Silver nanoparticles (AgNPs) and Ag + with a 1,10-phenanthroline complex induced mitochondrial apoptosis with caspase-3 and caspase-9 activation, decreased cell viability, and disrupted plasma and mitochondrial membrane integrity in experiments with hepatoblastoma HepG2 cells and A549 lung cancer cells [89,97,103]. AgNP-induced mitochondrial oxidative stress manifested as a decrease in reduced glutathione, mitochondrial uncoupling, ∆Ψ mito decline, a decrease in the antioxidant enzymes' activity, ATP synthesis stoppage, an increase in membrane leakage, ROS production, lipid peroxidation, and NADPH oxidase activity in experiments with BRL-3A hepatocytes, human keratinocytes HaCaT cells, human peripheral blood mononuclear cells, and mouse AML12 hepatocytes [102][103][104][105][106]. Microscopic studies found that silver-nanoparticle-exposed cells showed an abnormal size, cellular shrinkage, and irregular shape acquisition [104]. The interplay between AgNP-induced mitochondrial fission and mitophagy defects was shown in in vivo and in vitro experiments with human hepatocellular carcinoma HepG2 cells [107]. The features of the AgNP reaction with cellular and mitochondrial enzymes and structures allow for the further use of these particles in different areas, such as anticancer and antimicrobial therapy; the textiles, water treatment, and cosmetics industries; and biomedical research as antimicrobial, antifungal, antiviral, anti-inflammatory, and anti-angiogenic agents [105][106][107][108].
The crystal chemical radii of Ag + and K + ions are close in magnitude; therefore, Ag + can be transported through biological membranes via potassium channels. However, although Ag + has the highest affinity for the thiol groups of biomolecules and cellular structures, it cannot be classified as the most highly toxic agent. The general explanation is the ability of Ag + to react with Cl − anions and metallothioneins binding heavy metal ions. So, these interactions markedly decrease the active concentration of Ag + in the body's internal environment. Silver nanoparticles are devoid of these shortcomings, and they are able to reach critical thiol groups. Recent comparative studies of AgNPs and Ag + indicate this circumstance [109][110][111]. It was AgNPs that maximally reached and integrated into cellular structures, exerting a more substantial toxic effect on cells than Ag + . Understanding the factors determining the toxicity of AgNPs is very important for biomedical applications, particularly in cancer therapy. In this regard, modern studies of silver nanoparticles are very relevant in terms of the search for new anticancer and antimicrobial drugs. The Ag + ions that overcome these barriers damage DNA, inhibit respiratory chain complexes, and induce MPTP opening, which are accompanied by the calcium overload of mitochondria. Together, Ag + ions cause oxidative stress (Figure 1), which is accompanied by a decrease in ATP synthesis and the development of apoptotic processes ( Figure 2) leading to cell death. Undoubtedly, an excessive accumulation of silver in the body can contribute to developing diseases associated with metabolic disorders and lead to the degradation of body systems and the development of oncology. microbial drugs. The Ag + ions that overcome these barriers damage DNA, inhibit respiratory chain complexes, and induce MPTP opening, which are accompanied by the calcium overload of mitochondria. Together, Ag + ions cause oxidative stress (Figure 1), which is accompanied by a decrease in ATP synthesis and the development of apoptotic processes ( Figure 2) leading to cell death. Undoubtedly, an excessive accumulation of silver in the body can contribute to developing diseases associated with metabolic disorders and lead to the degradation of body systems and the development of oncology. General elements for toxic mechanisms of inducing mitochondrial oxidative stress via heavy metals. Heavy metals (Ag + , Hg 2+ , Cd 2+ , Pb 2+ , As 3+ , and Sb 3+ ) induce oxidative stress by blocking respiratory complexes with MPTP opening due to interacting with the thiol groups shown in the picture. Other metals (Tl + , Al 3+ , Ga 3+ , In 3+ , Cr 6+ , and U 6+ ) inactivate these thiol groups by indirectly oxidizing them due to activation of the production of oxygen radicals (ROS and H2O2). Black arrows show the metal's reaction with thiol groups (SH-). The MPTP induction by heavy metals is shown by the bold arrow. The induction of ROS and H2O2 is marked with a blue arrow. The ROS-induced oxidation of thiol groups is shown by red arrows. Pink arrows show the cytochrome C release into the intermembrane space due to the MPTP opening. Abbreviations: ADP, adenosine diphosphate; ANT, adenine nucleotide translocase; ATP, adenosine triphosphate; CI-CV, inner mitochondrial membrane complexes I-V; Cyt C, cytochrome C; ETC, electron transport chain; GSH, reduced glutathione; IMM, inner mitochondrial membrane; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; SH-, molecular thiol groups; ΔΨmito, inner membrane potential. General elements for toxic mechanisms of inducing mitochondrial oxidative stress via heavy metals. Heavy metals (Ag + , Hg 2+ , Cd 2+ , Pb 2+ , As 3+ , and Sb 3+ ) induce oxidative stress by blocking respiratory complexes with MPTP opening due to interacting with the thiol groups shown in the picture. Other metals (Tl + , Al 3+ , Ga 3+ , In 3+ , Cr 6+ , and U 6+ ) inactivate these thiol groups by indirectly oxidizing them due to activation of the production of oxygen radicals (ROS and H 2 O 2 ). Black arrows show the metal's reaction with thiol groups (SH-). The MPTP induction by heavy metals is shown by the bold arrow. The induction of ROS and H 2 O 2 is marked with a blue arrow. The ROS-induced oxidation of thiol groups is shown by red arrows. Pink arrows show the cytochrome c release into the intermembrane space due to the MPTP opening. Abbreviations: ADP, adenosine diphosphate; ANT, adenine nucleotide translocase; ATP, adenosine triphosphate; CI-CV, inner mitochondrial membrane complexes I-V; Cyt C, cytochrome C; ETC, electron transport chain; GSH, reduced glutathione; IMM, inner mitochondrial membrane; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; SH-, molecular thiol groups; ∆Ψ mito , inner membrane potential. Tl(I). Mitochondrial research. The inner mitochondrial membrane turned out to be noticeably permeable to Tl + ions [112,113]. We have shown that excess Tl + ions are removed from mitochondria when using a K + /H + exchanger, which is followed by the pumping out of incoming protons from the matrix [114]. In addition, Tl + does not inhibit mitochondrial respiratory enzymes [9,10,24,25]. This is why Tl + increased state 4 respiration and did not affect state 3 or 3U DNP respiration in energized RLM [9,24,25,[114][115][116][117]. As a similarly toxic heavy metal, Tl + increased the passive permeability of the IMM to univalent cations (K + , H + , and Na + ), and that was shown in experiments with non-energized RLM injected into a nitrate medium containing a mixture of nitrates of Tl + and one of the univalent cations [114]. Energized RLM added to the nitrate medium showed an increase in swelling General links in mitochondria-dependent pathways of intracellular apoptosis in by different-valence heavy metals. Heavy metals (Ag + , Tl + , Hg 2+ , Cd 2+ , Pb 2+ , As 3+ , Sb 3+ , Al In 3+ , Cr 6+ , and U 6+ ) induce mitochondrial permeability transition pore (MPTP) opening, w accompanied by cytochrome c release into the intermembrane space. As a result of this p caspases-3 and -9 are activated and trigger apoptosis induction. Some metals (Cd 2+ , As 3+ , A In 3+ ) damage DNA, resulting in bax and p53 activation that induces apoptotic processes. show MPTP induction. Bold vertical arrows show the sequence of events associated wit chondria during the induction of intracellular apoptosis by heavy metals. Apoptosis in pathways associated with DNA damage are indicated by horizontal arrows. Abbreviations: cytochrome C; MPTP, mitochondrial permeability transition pore.

Tl(I). Mitochondrial research.
The inner mitochondrial membrane turned ou noticeably permeable to Tl + ions [112,113]. We have shown that excess Tl + ions moved from mitochondria when using a K + /H + exchanger, which is followed pumping out of incoming protons from the matrix [114]. In addition, Tl + does not mitochondrial respiratory enzymes [9,10,24,25]. This is why Tl + increased state 4 r tion and did not affect state 3 or 3UDNP respiration in energized RLM [9,24,25,114 As a similarly toxic heavy metal, Tl + increased the passive permeability of the IM univalent cations (K + , H + , and Na + ), and that was shown in experiments non-energized RLM injected into a nitrate medium containing a mixture of nitrate and one of the univalent cations [114]. Energized RLM added to the nitrate m showed an increase in swelling and state 4 respiration as well as some decline in and 3UDNP respiration and the inner mitochondrial membrane potential (ΔΨmito) [1 Figure 2. General links in mitochondria-dependent pathways of intracellular apoptosis induction by different-valence heavy metals. Heavy metals (Ag + , Tl + , Hg 2+ , Cd 2+ , Pb 2+ , As 3+ , Sb 3+ , Al 3+ , Ga 3+ , In 3+ , Cr 6+ , and U 6+ ) induce mitochondrial permeability transition pore (MPTP) opening, which is accompanied by cytochrome c release into the intermembrane space. As a result of this process, caspases-3 and -9 are activated and trigger apoptosis induction. Some metals (Cd 2+ , As 3+ , Al 3+ , and In 3+ ) damage DNA, resulting in bax and p53 activation that induces apoptotic processes. Arrows show MPTP induction. Bold vertical arrows show the sequence of events associated with mitochondria during the induction of intracellular apoptosis by heavy metals. Apoptosis induction pathways associated with DNA damage are indicated by horizontal arrows. Abbreviations: Cyt. C, cytochrome C; MPTP, mitochondrial permeability transition pore.
It has been previously postulated that thallium(I) toxicity is due to the ability of Tl + to easily penetrate the IMM and uncouple oxidative phosphorylation [24,112]. Tl + ions transport into energized mitochondria using both the mitochondrial ATP-sensitive potassium channel (mitoK ATP ) and the mitochondrial BK-type Ca 2+ -activated potassium channel (mitoK Ca ) [118,119]. Massive mitochondrial swelling was found in in vivo experiments using thallium(I) salts [15,25,120]. One reason for this swelling may be MPTP opening in the inner mitochondrial membrane. We found that such phenomena as mitochondrial swelling, state 3 and 3U DNP decrease, and ∆Ψ mito decline were markedly increased in experiments with energized calcium-loaded mitochondria [121,122]. These effects were completely eliminated or markedly attenuated by MPTP inhibitors (ADP, CsA, bongkrekic acid (BKA), and NEM), mitochondrial Ca 2+ uniporter blockers (RR, Y 3+ , La 3+ , Sr 2+ , and Mn 2+ ), or a Ca 2+ -chelator (EGTA) [115,121,[123][124][125]. Tl + -induced MPTP opening is possible only in the case of calcium-loaded mitochondria, while such loading is not required in similar experiments with heavy bivalent metals. The inhibition of mitoK ATP and mitoK Ca decreased the matrix calcium retention and accelerated the Tl + -induced MPTP opening in calcium-loaded rat liver mitochondria [126]. The substrate specificity effect [127] was found in succinate-energized mitochondria, which had more resistance to calcium loading than those energized by CI substrates [121]. Tl + -induced MPTP opening is dependent on the adenine nucleotide translocase (ANT) conformation [124]. The relationship between the ANT conformation and MPTP opening in the inner membrane was substantiated previously in detail [128,129]. The pore-opening phenomena were more noticeable in fixing the ANT c-conformation induced by thiol reagents (phenylarsine oxide (PAO), 4,4diisothiocyanostilbene-2,2 -disulfonate (DIDS), and high NEM), thiol oxidizers (tert-butyl hydroperoxide (tBHP), diamide (Diam)), and carboxyatractyloside (CAT) [124,130]. On the contrary, the manifestation of these phenomena noticeably decreased or completely inhibited the stabilization of the ANT m-conformation induced by reagents (ADP, low NEM, BKA, and eosin-5-maleimide (EMA)) and low mersalyl [9,124,131]. Additionally, Tl + -induced MPTP opening was recently found to be dependent on the activity of cysteine and lysine residues in the inner membrane proteins [131,132].
However, it is very likely that the Tl + -induced decrease in reduced glutathione in the matrix was due to our hypothesized reversible oxidation of Tl + to Tl 3+ near the centers of the generation of ROS in the respiratory chain [9]. It should be noted that thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc [21,143].
Tl + and K + cations (as well as Ag + ) have similar crystal chemical radii. For this reason, Tl + ions enter cells through potassium channels. However, Tl + (unlike Ag + and other heavy metals) has a minimal affinity for thiol groups. For this reason, Tl + really does not bind to intracellular metallothioneins and quickly penetrates cells, disrupting potassiumdependent cell processes. This distinguishing Tl feature is especially true of the toxic effects of Tl + ions on neurons, cardiomyocytes, and kidney cells. With a more prolonged exposure to the body, Tl + reduces the concentration of reduced glutathione while increasing ROS production in cells. Oxidative stress ( Figure 1) and apoptotic processes ( Figure 2) develop due to damage to cellular and mitochondrial membranes, decreased ATP synthesis, and the subsequent induced MPTP opening and calcium overload of cells and mitochondria. Such features of the chemical behavior of Tl + do not allow this metal to be rapidly removed from the body. Future research should focus on finding effective Tl + binding reagents for the subsequent elimination of this metal from the body to treat thallium-induced damage to the nervous system, kidneys, and hairline [9].
Hg(II). Mitochondrial research. Hg 2+ was shown to induce oxidative stress in experiments with RLM energized by CI and CII substrates [42,[144][145][146]. There was a state 3 and 3U DNP respiration decrease, ∆Ψ mito decline, mitochondrial swelling, a state 4 respiration increase, a mitochondrial membrane fluidity increase, cytochrome c release, and MPTP-induced mitochondrial ATP depletion. So, Hg 2+ to Cys binding mediates multiple Hg 2+ toxic impacts, especially the inhibition of enzymes and other proteins containing free Cys residues causing oxidative stress [147]. These unfavorable effects were prevented by MPTP inhibitors (ADP, CsA, Mg 2+ , and BKA) and Ru360 (an MCU blocker). F 1 F O -ATPase activity was promoted by micromolar Hg 2+ , and respiration in state 4 was inhibited in swine heart mitochondria energized by NADH but not succinate [148]. Hg 2+ and MeHg significantly declined mitochondrial viability and increased the H 2 O 2 and ROS production, lipid peroxidation, and glutathione oxidation in mouse brain mitochondria [149]. Quercetin and catalase prevented these effects of Hg 2+ . Hg 2+ resulted in swelling, a Ca 2+ efflux, and an ROS production increase in experiments with RKM [150]. The calcium load of these mitochondria additionally increased swelling and induced ∆Ψ mito decline. Tamoxifen and CsA inhibited these in vitro effects of Hg 2+ . Ca 2+ uptake and Ca 2+ -induced ∆Ψ mito decline were inhibited in mitochondria isolated from the kidneys of rats injected with mercury chloride plus tamoxifen [150]. Tamoxifen was concluded to be an inhibitor of Hg 2+ -induced MPTP [150].
Hg(II). Cell research. MeHg similarly to Hg 2+ induces mitochondrial apoptosis and oxidative stress, mediating an increase in ROS production, a decrease in cell antioxidant defense, and cytochrome c release from mitochondria [151,152]. Hg 2+ induced apoptosis with caspase-3 activation, a cell viability decrease, mitochondrial integrity decline, and intracellular ATP depletion. There was oxidative stress manifested as a decline in ∆Ψ mito , cytochrome c release, an increase in ROS generation, an increase in lipid peroxidation, a decrease in ATP and reduced glutathione, and a decrease in thioredoxin reductase and glutathione peroxidase activity in human hepatoma HepG2 cells, human neuroblastoma SH-SY5Y cells, human gingival fibroblasts, hamster pancreatic HIT-T15 β-cells, normal rat kidney cells, rat ascites hepatoma AS-30D cells, and human T cells and leukocytes [153][154][155][156][157][158][159][160][161][162]. N-acetylcysteine reversed some of these effects of Hg 2+ . Experiments with Hg 2+ -and MeHg-treated fibroblast zebrafish ZF4 cells found a decrease in ATP production and mitochondrial respiration in the basal, 3, and 3U FCCP states [163]. However, the proton leak and non-mitochondrial respiration were not changed there [163]. Hg 2+ induced a cell viability decrease, ∆Ψ mito decline, and an ROS production increase in experiments with five Tetrahymena species; however, more ingested MeHg disrupted the membrane integrity [164].
The Hg 2+ -and MeHg-induced apoptosis in HepG2 cells can be due to Hg 2+ , and MeHg inhibited the respiration, increased the permeability, and blocked the essential thiol proteins of mitochondrial membranes [165]. The inner membrane protein thiols were dose-dependently decreased by Hg 2+ in swine heart mitochondria [148]. However, these Hg 2+ -induced effects were reversed by DTT due to the latter's interaction with the Cys residues of respiratory complexes [148]. A Fenton-type reaction may be the cause of the Hg 2+ -induced oxidative stress and ROS production in NRK-52E cells due to the binding of Hg 2+ to intracellular thiols (proteins or glutathione) [166]. Hg 2+ increased free Ca 2+ levels in canine kidney cells due to both extracellular Ca 2+ influx (protein kinase C-regulated) and the phospholipase C-activated Ca 2+ release from endoplasmic reticulum (ER) [167]. MeHginduced neurotoxicity in primary rat cortical neurons stimulated Ca 2+ release, increasing from the ER simultaneously with MPTP opening and ∆Ψ mito decline [168].
Hg 2+ penetrates cells and mitochondria via calcium-transporting mechanisms. Hg 2+ ions induce oxidative stress ( Figure 1) and apoptotic processes ( Figure 2) in cells and mitochondria, having a noticeable affinity for thiol groups. This stress manifests in a marked decrease in CI and CII activities, a decline in ∆Ψ mito , the induction of MPTP opening, and a decrease in ATP and GSH synthesis. At the same time, the calcium overload of cells and mitochondria sharply increases, ROS production increases, and the reduced GSH concentration decreases. At higher levels, Hg 2+ damages renal nephrons. The main toxic manifestations in this case can be considered the induction of MPTP opening and the blocking of critical thiol groups by Hg 2+ ions because the MPTP inhibitors (ADP, CsA, and tamoxifen) and reducers of these groups (DTT and NAC), along with calcium transport blockers (RR and Ru360), eliminated the toxic effects of Hg 2+ .
Cd(II). Mitochondrial research. Studies of the cadmium effects on various characteristics of isolated mitochondria were conducted. Cd 2+ in succinate-energized RLM induced ∆Ψ mito decline; iron mobilization; a decrease in state 3 and 3U DNP respiration, ATP content, potential dependent 137 Cs + uptake, and protein thiol levels; and an increase in state 4 0 respiration, IMM proton permeability, and H 2 O 2 production; but Cd 2+ slowly affected RLM succinate dehydrogenase activity [48,54,144,146,169]. Cd 2+ (similarly Hg 2+ ) increased the inner membrane passive permeability to H + and K + ions and showed ∆Ψ mito collapse, a decrease in 3U DNP respiration, and an increase in K + mitochondrial uptake and state 4 0 respiration in RHM energized by CI and CII substrates [42,46,47]. Cd 2+ resulted in respiration inhibition in 3 and 3U FCCP states, ∆Ψ mito decline, cytochrome c release, Ca 2+ uptake inhibition, and a Ca 2+ release and H 2 O 2 generation increase in isolated rat kidney mitochondria [39,55,170]. DTT and CsA prevented the Cd 2+ -induced Ca 2+ efflux and ∆Ψ mito decline in energized rat kidney mitochondria [171]. Quinine inhibited the contraction of succinate-energized RKM preswollen in a KCl medium containing Cd 2+ [39].
After reacting with the outer and inner thiol groups of the inner membrane, Cd 2+ and Hg 2+ then penetrate into mitochondria and cause the release of cytochrome c, the opening of MPTP, and the inhibition of CI and CII [42]. After penetrating into the matrix through the mitochondrial Ca 2+ uniporter, Cd 2+ was assumed to induce mitochondria osmotic swelling and the cytochrome c release due to the activation of aquaporin H 2 O channels because mitoplast swelling was blocked by Ag + (a potent aquaporin blocker) [172]. The Cd 2+ -induced swelling, ∆Ψ mito decline, state 4 0 increase, IMM fluidity increase, and lipid peroxidation were decreased in the presence of MPTP inhibitors (ADP, CsA, Mg 2+ , DTT, NEM, RR, and Ru360) in experiments with succinate-energized RLM [39,44,48,146,173]. Moreover, the maximum effect was achieved with the simultaneous presence of some of these inhibitors in salt media containing NH 4 NO 3 , KCl, or sucrose [44]. Atractyloside prevented the contraction of succinate-energized RLM preswollen in a NH 4 NO 3 medium containing 15 µM Cd 2+ , oligomycin, and rotenone [44]. CsA partly inhibited the Cd 2+induced state 4 0 increase and state 3U DNP decrease in succinate-energized RLM [44]. Thus, Cd 2+ -induced mitochondrial dysfunction appears to be associated with the opening of ADP-and CsA-dependent MPT pores in the inner membrane [44]. Cd 2+ resulted in MPTP opening, ∆Ψ mito decline, cytochrome c release, decreased state 3 respiration, and increased state 4 0 respiration. These Cd 2+ effects were completely suppressed by Bcl-xL, RR, and BKA but not by CsA in experiments with succinate-energized mouse liver mitochondria [174]. This Cd 2+ -induced MPTP opening was supposed to be the reason for the Cd 2+ interaction with ANT thiol groups [174].
We have recently been shown that Mg 2+ with the ADP inhibition of the state 4 0 increase, induced by high Cd 2+ with RR, was not changed in experiments with succinate-energized RLM added to a K-acetate medium containing both a mitoK ATP inducer (diazoxide) and the channel inhibitors (5-hydroxydecanoate (5-HD) and glibenclamide) [146]. However, the Cd 2+ -induced swelling of RLM-energized glutamate with malate increased in a KCl medium containing the mitoK ATP inhibitor ATP. This effect was slightly prevented by diazoxide and, in contrast, was markedly potentiated by 5-HD [146]. So, one can conclude that the above inhibition of the Cd 2+ -induced state 4 0 increase is probably due to the mitoK + uniporter blocking by ATP with Mg 2+ but not the MPTP opening. On the contrary, the Cd 2+ -induced swelling increase in the KCl medium containing mitoK ATP inhibitors (ATP and 5-HD) is probably due to the greater MPTP opening when the mitoK ATP channels are closed. This result is in good agreement with the previous assumption that the probability of the MPT pore opening in the inner membrane increases when the mitoK ATP channels are closed [175][176][177].
Research on energized RLM found that the state 4 0 respiration increase and the state 3U DNP respiration decrease induced by Cd 2+ with Ca 2+ were visibly eliminated in the presence of ADP with CsA [54,178]. However, Cd 2+ -induced state 4 0 respiration increased even more after the administration of DTT but not EGTA, which was injected after high Cd 2+ [54,144,178]. Ca 2+ with Pi or NEM alone markedly increased the Cd 2+ -induced passive proton permeability of the inner membrane. In this case, preswollen RLM (after the succinate addition) showed some contraction in a NH 4 NO 3 medium containing low NEM (10-100 µM) or Pi alone but not Ca 2+ with/without Pi or high NEM (1 mM). This contraction became much more potent after the DTT addition to the medium containing NEM with rotenone, and the succinate-energized mitochondria visibly contracted (like the control Cd 2+ -free experiments), even in the presence of 1 mM NEM (except for in the experiments where Cd 2+ , Ca 2+ , and Pi were all present) [44,54,178]. This research concluded that Cd 2+ -induced mitochondrial dysfunction is due to the fact that this metal acts as a thiol and Me 2+ binding site reagent [54]. CsA inhibited an additional Cd 2+induced increase in K + transport in succinate-energized RLM in the presence of ruthenium red [178]. Experiments with cadmium metallothionein in vivo and Cd 2+ in vitro found renal cortical mitochondria swelling and respiratory function decline (a decrease in state 3 and the respiratory control ratio (RCR)) that indicates both electron transfer and oxidative phosphorylation inhibition [179].
Cd(II). Cell research. Cd 2+ transports into cells by using ion channels, special carriers, Ca 2+ /calmodlin-dependent protein kinase II (CaMK-II), and ATP-hydrolyzing pumps, whereas Cd 2+ -protein complexes penetrate the membrane through receptor-mediated endocytosis of the cadmium-metallothionein complexes but not N-and L-type Ca 2+ channels, which are closed at the resting membrane potential [180][181][182]. In addition, the mitochondrial Ca 2+ uniporter is involved in the Cd 2+ transport into mitochondria [180].
Being a physiological secondary messenger, Ca 2+ and ROS control Ca 2+ -dependent and redox-sensitive molecular processes that determine cell function and fate. Ca 2+ and ROS are known to activate cell death effectors (caspases, ceramides, calpains, and p38), to irreversibly damage mitochondria and the endoplasmic reticulum, and to modulate biosynthesis metallothioneins and Bcl-2 proteins [183]. It is the Ca 2+ and ROS increase that is directly affected by the Cd 2+ impact in experiments in vitro and in vivo [183]. Cd 2+ produced an increase in the cytoplasm Ca 2+ concentration in canine kidney cells, rat primary astrocytes, rat primary cerebral cortical neurons, yeast cells, mouse skin fibroblasts, and rat hepatocytes [184][185][186][187][188][189]. Cd 2+ induced the release of the stored Ca 2+ from the endoplasmic reticulum and mitochondria in canine kidney cells and NIH 3T3 cells [184,190]. Cd 2+ decreased agonist-induced ER calcium signals and sarcoplasmic-ER calcium ATPases activity in NIH 3T3 cells [190].
Cd 2+ in normal human lung MRC-5 fibroblasts caused caspase-independent apoptosis mediated by ROS scavengers (NAC and mannitol) to indicate the crucial role of ROS in apoptogenic Cd-activated apoptosis. The Cd 2+ -induced apoptosis was partially or completely abrogated by CI and CV inhibitors (rotenone and oligomycin A) or MPTP inhibitors (CsA and aristolochic acid) [212].
Cd 2+ actively interacts with the IMM thiol groups of cells and mitochondria. The result of this interaction is the induction of apoptosis ( Figure 2) and oxidative stress (Figure 1) manifesting in increased ion transport through IMM, the inhibition of mitochondrial respiratory complexes (CI-CIII), a decrease in ATP synthesis, the calcium overload of mitochondria, an increase in ROS production, a decline in ∆Ψ mito , and the induction of the MPTP opening. At a higher body level, Cd 2+ damages target organ cells (nervous tissue and liver). Considering the closeness of Cd 2+ and Ca 2+ radii, it should be noted that the toxic effects of Cd 2+ increase significantly under conditions of the calcium overload of cells and mitochondria. The above Cd 2+ effects, regardless of the presence of calcium, were markedly attenuated in the presence of calcium transport blockers and competitors (RR, Ru360, Sr 2+ , and Mn 2+ ), MPTP inhibitors (CsA and BKA), and thiol-reducing agents (NAC and DTT). Therefore, the main three toxic Cd 2+ manifestations leading to oxidative stress and apoptosis can be considered the Cd 2+ -induced calcium overload of mitochondria, the blocking of critical thiol groups, and the opening of MPTP. Cadmium toxicity processes can involve mitochondrial s-glutathionylation. Cd 2+ disturbed the redox balance of mitochondria due to an ROS production increase, which enhanced the S-glutathionylation of mitofusin 2 and damaged the membranes binding ER with subsequent neuronal necroptosis [222].
Pb(II). Mitochondrial research. Experiments with mitochondria showed that Pb 2+induced mitochondrial oxidative stress manifested in the inhibition of CII-CIV as well as in an increase in ROS production and lipid peroxidation, ATP consumption, and glutathione oxidation [223,224]. In parallel, Pb 2+ decreased the activity of mitochondrial antioxidant system enzymes (GPX, SOD, catalase, and GSH) [224]. At the same time, Pb 2+ induced MPTP opening that manifested in RLM swelling, a state 3 respiration decrease, ∆Ψ mito decline, an IMM fluidity decrease, proton influx in the matrix, and K + and cytochrome c release. The Pb 2+ -induced swelling visibly declined in the presence of known MPTP inhibitors (CsA and ADP), chelators (EGTA and EDTA), and a mitochondrial Ca 2+ uniporter inhibitor (RR) [71,223,224].
Pb(II). Cell research. Pb 2+ induced apoptosis, including the activation of caspase-3 and the expression of the apoptotic-inducing factors (Bax, Bcl2, and Bcl-2), MPTP opening, and oxidative stress that manifested as mitochondrial function oxidative damage, cytochrome c release, and a decrease in cell viability, reduced glutathione, and ATP production as well as an ROS production increase, ∆Ψ mito decline, Ca 2+ release from the matrix, and a decline in the mitochondrial Ca 2+ uniporter expression in human neuroblastoma SH-SY5Y cells, rat proximal tubular cells, Siberian tiger fibroblasts, rat insulinoma β-cells, PC12 cells, and adult rat hepatic stem cells [225][226][227][228][229][230][231][232]. These effects of Pb 2+ were eliminated by MPTP inhibitors (CsA, DIDS, and BKA), which may indicate the participation of Cyp-D and ANT in Pb 2+ -induced MPTP opening. It was concluded that the mitochondrial Ca 2+ influx regulation in neurons is mediated by the Pb 2+ -induced oxidative stress response.
The mechanism of Pb 2+ neurotoxicity is associated with this cation ability to replace or compete with important biogenic cations (Ca 2+ , Fe 2+ , and Zn 2+ ) [233]. Pb 2+ -induced oxidative stress manifested in mitochondrial uncoupling; the inhibition of CI and CIII; a decrease in the matrix reduced glutathione; ∆Ψ mito ; mitochondrial O 2 consumption; an increase in the IMM ion permeability, ROS production, and lipid peroxidation; and a decrease in cytoplasm ATP due to an energy consumption imbalance [233]. All these toxic Pb 2+ effects led to the displacement of Ca 2+ from intracellular compartments, the opening of MPTP, and the induction of apoptotic and necrotic changes in neurons and cells [233]. Pb 2+ induced a reduction in glucose-stimulated insulin secretion in experiments with rat insulinoma β-cells [230].
The toxic actions of Cd 2+ , Hg 2+ , and Pb 2+ were studied in vitro on pancreatic β-cells from CD-1 mice [234]. These metals resulted in oxidative stress manifesting as a decrease in ∆Ψ mito , ATP production, CI-CIV activity, and oxygen consumption rates with a parallel increase in cell lactate production, mitochondrial succinate-supported swelling, and IMM permeability to K + and H + ions as well as an increase in membrane fluidity and a decrease in saturated/unsaturated fatty acid ratios [234]. Pb 2+ damaged liver mitochondrial and nuclei structures and activated mitochondrial apoptosis through caspase-3 and caspase-9 signaling pathways in experiments with Pb-poisoned chickens [235]. The independence of the toxic mechanisms of Cd and Pb was proven in experiments with rats exposed to Cd acetate [211]. Pb 2+ alone resulted in a decrease in liver superoxide dismutase (SOD) activity; however, catalase activity and glutathione (GSH) or thiobarbituric acid content were unchanged. Cd 2+ with/without Pb 2+ resulted in rat liver oxidative stress because a decrease in SOD activity and GSH content and an increase in catalase activity and thiobarbituric acid content were found.
A distinctive feature of Pb 2+ ions is their ability to compete with Ca 2+ for calcium transport systems of the plasma membrane. However, by entering the mitochondria, Pb 2+ inhibits Ca 2+ uptake and displaces Ca 2+ and K + from the matrix. The main targets of Pb 2+ are the cells of the nervous system, pancreas, and liver. Given the ability of lead to displace calcium from cells, osteoblasts may be another reason for Pb 2+ toxic effects leading to bone demineralization. After penetrating mitochondria, Pb 2+ induces oxidative stress ( Figure 1) and apoptotic processes (Figure 2), interacting with thiol groups and displacing essential biogenic cations (Ca 2+ , Fe 2+ , and Zn 2+ ) from mitochondrial structures. Such oxidative stress is characterized by decreased CII-CIV activity, MPTP opening, ∆Ψ mito decline, decreased ATP consumption, increased ROS production and lipid peroxidation, and glutathione oxidation, reducing the activity of mitochondrial antioxidant system enzymes. That is why Pb 2+ toxicity visibly declines in the presence of known MPTP inhibitors (ADP, CsA, BKA, and DIDS), chelators (EGTA and EDTA), antioxidants (catalase), and a mitochondrial Ca 2+ uniporter inhibitor (RR). Since lead enters the environment due to various technological processes, it has become essential to develop ways to protect the body from the toxic manifestations of lead compounds and remove them from the body. Pb 2+ can accumulate in astrocytes and have a pronounced poisonous effect, disrupting glutamate metabolism [236]. Additionally, the activation of S-glutathionylation processes was found because of the enhanced expression of glutathione-protein complexes after Pb treatment.
The accumulation of heavy metals (Hg, Cd, Pb, Cr, U, and As) in living organisms leads to toxic damage to different organs and tissues. These heavy metals disturb various biological functions, including proliferation, differentiation, damage repair, and apoptosis induction [237][238][239]. At the same time, the mechanisms of toxicity induction often have common patterns directly related to the occurrence of mitochondrial oxidative stress. An ROS production increase, antioxidant defense reduction, enzyme inactivation, MPTP opening, calcium overload, and ∆Ψ mito decline belong to these patterns. The toxic effects of these metals are multilevel and may play a role in the development of cancer, autoimmune diseases, diabetes, toxicogenomic disorders, Parkinson's disease, and other metabolic disorders.

Al(III), Ga(III), and In(III). Mitochondrial research.
Al 3+ decreases the Ca 2+ uptake in ER, induces the release of Ca 2+ from the matrix, and potently inhibits Ca 2+ -ATPase activity in RLM that can result in intracellular calcium overload [240]. Aluminum nanoparticles (AlNPs), more so than Al 3+ , induced oxidative stress that manifested as an increase in mitochondrial swelling, ROS production, and lipid peroxidation, as well as CIII inhibition, ∆Ψ mito decline, and cytochrome c release, in experiments with isolated rat brain mitochondria [241]. Al 3+ plus tyramine induced MPTP opening in Ca 2+ -loaded RLM that manifested as mitochondrial swelling, an ROS and H 2 O 2 production increase, mitochondria morphological alterations, and the oxidation of pyridine nucleotides and mitochondrial thiols [242]. These effects were completely eliminated by MPTP inhibitors (CsA, DTT, and NEM) and partly eliminated by catalase. The opening of this pore requires the simultaneous oxidation of thiol groups on both sides of the inner mitochondrial membrane [242].
In 3+ in experiments with succinate-energized RLM induced mitochondrial swelling, ∆Ψ mito decline, ROS production, a proton permeability increase, and electron transition inhibition [243]. In 3+ -induced MPTP was due to the inhibition of the inner membrane proton channels and the stimulation of mitochondrial oxidative stress, resulting in a 15-20% decrease in mitochondrial respiration in states 4, 3, and 3U DNP [243]. The In 3+ -induced swelling, ∆Ψ mito decline, and proton permeability increase were markedly eliminated by MPTP inhibitors (ADP and CsA), chelating agents (EGTA and EDTA), and ruthenium red but not by the thiol reagent DTT.  [244]. Both Al 3+ and aluminum compounds target body tissues resulting in bone mineralization impairment, hemoglobin synthesis impairment, and cardiovascular and neurological disorders [244]. Al 3+ ions additionally increased glutamate-induced intracellular Ca 2+ overload in rat hippocampal organotypic cultures [245]. Al 3+ resulted in an intracellular Ca 2+ increase followed by the inhibition of protein kinase B phosphorylation, resulting in cell morphology impairment in PC12 cells [246].

Al(III), Ga(III), and In(III
Al 3+ -induced apoptosis accompanied by the activation of caspases-9, -8, and -3 and oxidative stress manifested as a cell viability decrease, cytochrome c release, and ∆Ψ mito decline in experiments with rat osteoblasts [247,248]. Al 3+ induced an ROS generation increase and mitochondrial damage, resulting in a decrease in SOD and catalase activities and ATP synthesis in hippocampal neuronal cells and human HepG2 cells [249][250][251]. These effects were attenuated by chlorogenic acid [249]. Al 3+ -induced oxidative stress showed toxic effects that manifested as cell viability collapse, ∆Ψ mito decline, an increase in ROS production, and a decrease in oxygen consumption and mitochondria enzyme activity in experiments with rat nerve cells and human peripheral blood mononuclear cells [252,253]. Experiments with rats fed with AlCl 3 via drinking water showed that Al 3+ caused a reduction in the cytoplasm ATP level, a decrease in CI-CIV activity, and disturbance to mitochondrial DNA transcripts, which was followed by the inhibition of the mRNA expression of NADH dehydrogenases 1 and 2, cytochrome b, cytochrome c oxidase subunits 1 and 3, and ATP synthase [254]. By violating the mitochondrial energy metabolism, AlCl 3 caused an increase in important liver aspartate and alanine aminotransferases and histopathological lesions. Aluminum nanoparticles (AlNPs), more so than Al 3+ , induced apoptosis, a cellular viability decrease, a DNA damage increase, mitochondrial dysfunction, and oxidative stress, as well as a decrease in ∆Ψ mito and reduced glutathione, in experiments with human hepatic HepG2 cells and differentiated HepaRG cells [255].
Ga 3+ ions transport into cells via the use of cell Fe 3+ transport systems [256]. A decrease in Ga 3+ -induced cellular iron uptake and intracellular iron homeostasis disruption resulted in mitochondrial function inhibition due to an intracellular ROS production increase and a reduced glutathione decrease as well as iron-containing proteins being targeted in the electronic transport chain [256]. Ga 3+ triggers apoptosis by Bax or p53 activation, which was followed by translocation to mitochondria, which results in ∆Ψ mito loss, an ROS production increase, and cytochrome c release into the intermembrane space and the cytoplasm [256].
Indium(III) compounds were found to induce a cell viability decrease, a lipid peroxidation and ROS production increase, reduced glutathione depletion, an SOD activity decrease, ∆Ψ mito decline, and oxidative stress damage as well as the expression of apoptotic genes (p53, bax, bcl-2, caspase-3, and caspase-9), oxidative DNA damage, and the formation of condensed chromosomal bodies in experiments in vitro with human lung epithelial A549 cells, lung-derived epithelial (LA-4) cells, human bronchial epithelial BEAS-2B cells, human embryonic kidney 293T cells, and mouse monocyte macrophage RAW 264.7 cells [243,[257][258][259][260][261]. In 3+ in experiments with human GM5565 skin fibroblasts affected mitochondrial morphology, and organelles became fragmented with a dotted geometry, which may indicate organelle aggregation caused by increased ROS production [262]. Indium oxide nanoparticles were found to have an acute toxic influence on epithelial 16HBE and macrophage (RAW264.7) cells in experiments with rats subjected to particle inhalation [263]. Damage to mitochondria, a rough endoplasmic reticulum, and a cell viability decrease were found.
These trivalent cations (Al 3+ , Ga 3+ , and In 3+ ) enter cells using Fe 3+ transport systems, which was followed by ROS production with lipid and protein oxidation and iron homeostasis disruption and a reduced glutathione decrease. As a result, damage to iron-containing proteins takes place in the electronic transport chain. There is an entirely different mechanism for inducing both oxidative stress ( Figure 1) and apoptosis (Figure 2). The free radical oxidation of thiol groups caused by these metals leads to decreased CI-CIV activity, ATP production, and ∆Ψ mito decline as well as decreased SOD and catalase activities. The expression of apoptotic genes and oxidative DNA damage accompanies apoptosis induction. Of course, these trivalent metals can be attributed to toxicants disrupting iron and calcium metabolism. This circumstance can harm the hematopoietic system and the nervous system cells. However, accumulating these metals in the body can aggravate diseases associated with the degradation of the nervous and hematopoietic systems and the synthesis of hemoglobin and other iron-containing proteins.
As(III). Mitochondrial research. As(III) (As 2 O 3 or AsO 2 − ) induced a decrease in CI and CII activities, ∆Ψ mito , and glutathione content, as well as an increase in ROS production, lipid peroxidation, cytochrome c release, and swelling, in isolated RLM or mouse liver mitochondria [264][265][266][267]. Some of these As 3+ toxic effects were increased by pyruvate [264] and attenuated by alpha lipoic and ellagic acids [265,268]. AsO 2 − induced an H 2 O 2 production increase in isolated RHM [269]. As 2 O 3 -induced MPTP opening was accompanied by an increase in mitochondrial swelling and Ca 2+ -induced Ca 2+ release, cytochrome c release from the matrix in the inner membrane space, and ∆Ψ mito decline in succinate-energized RLM [270,271]. However, Ca 2+ is required for MPTP opening. CsA, NEM, or RR inhibited MPTP. As 3+ did not affect the mitochondrial GSH content in RLM [271]. RLM takes up H 2 AsO 4 − via the Pi-dependent pathway and can reduce As 5+ to As 3+ in participating thioredoxin reductase and reduced glutathione, which is followed by the extrusion of As 3+ from mitochondria [272]. This process is more effective with CI substrates than succinate or ADP but is abolished by electron transport inhibitors, uncouplers, ATP synthase inhibitors, and Pi-transport inhibitors.
As(III). Cell research. The available literature analysis makes it possible to attribute As(III) chemical forms (As 2 O 3 , AsO 2 − ) to mild thiol reagents. This is the reason for their widespread use in ancient Chinese medicine and in the treatment of certain cancers. Arsenic (As 2 S 2 , As 2 O 3 , NaAsO 2 , and Na 2 HAsO 4 ) and mercury (HgS, HgCl 2 , and MeHg) are widely used in oriental medicine [273]. It was found that the AsO 2 − -induced superoxide increase in human myeloid leukemia U937 cells has two contrary pathways: the first leads to the promotion of Nrf2 signaling followed by GSH biosynthesis; the second results in DNA damage and MPTP opening with rapid and massive apoptotic cell death [274,275]. As(III) (AsO 2 − and As 2 O 3 ) increased the intracellular Ca 2+ concentration and mitochondrial dysfunction due to Ca 2+ mobilizing from the ER in human myeloid leukemia U937 cells, human bronchial epithelial HBE cells, and human umbilical and bone marrow mesenchymal stem cells [276][277][278].
N-acetyl-L-cysteine, L-ascorbic acid, and selenite (SeO 3 2− ) prevented AsO 2 − -induced apoptosis, oxidative stress, a cell viability decrease, mitochondrial disfunction, cytochrome c release, and an ROS production increase in experiments with L02 hepatocytes, mouse oligodendrocyte precursor cells, human myeloid leukemia U937 cells, and acute promyelocytic leukemia NB4 cells [279,[299][300][301]. The effect of SeO 3 2− can be caused by the inhibition of AsO 2 − transport in NB4 cells [301]. Pterostilbene or tert-butylhydroquinone activating the Nrf2 pathway alleviated similar AsO 2 − -induced effects in human HaCaT keratinocytes, mouse epidermal JB6 cells, and human epithelial HaCaT cells [302,303]. An increased glutathione biosynthesis capacity prevented As 3+ -induced apoptosis due to a decrease in caspase activation and cytochrome c release in the cytoplasm in mouse liver hepatoma Hepa-1c1c7 cells [280]. Autophagy can inhibit As 2 O 3 -induced apoptosis in HL60 cells in the initiation stage [304]. Metallothionein or methyl donors (betaine, methionine, and folic acid) alleviated the As 3+ -induced ∆Ψ mito decline and ROS production increase in PC12 cells or isolated rat hepatocytes [289,290]. The ferroptosis inhibitor Fer-1 attenuated the similar toxic effects of As 2 O 3 on rat cardiomyocyte H9c2 cells [291]. Experiments with rats co-exposed to NaAsO 2 and NaF showed oxidative stress and autophagy in myocardial tissue and cells [287]. As 3+ -induced oxidative stress and the apoptosis mitochondrial pathway were alleviated in experiments with the glutathione treatment of chronic arsenic-exposed mice [305].
Arsenic nanoparticles induced by apoptosis, mitochondrial swelling, ∆Ψ mito decline, a glutathione decrease, an ROS production increase, and membrane integrity were disturbed in isolated rat hepatocytes [306].
Sb(III). Mitochondrial and cell research. Experiments with Sb 3+ -treated mitochondria isolated from A549 cells showed an increase in SOD activity and a decrease in CI and CIII activities, glutathione peroxidase, glutathione reductase, and thioredoxin reductase [307]. The compounds and complexes of pentavalent and trivalent antimony with potassium antimonyl tartrate are used to treat leishmaniasis caused by protozoan parasites [308][309][310]. Sb(III) (Sb 2 O 3 and SbCl 3 ) induced apoptosis and cell viability loss; an increase in ROS production; and a decrease in the cytoplasm glutathione level, ∆Ψ mito , and ATP content in human lung adenocarcinoma A549 cells, human embryonic kidney HEK-293 cells, and CCRF-CEM line cells [307,311,312]. Potassium antimonyl tartrate (K 2 [SbC 2 H 2 (O) 2 (COO) 2 ] 2 ) and As 2 O 3 had similar effects in experiments with human lymphoid tumoral cells and human myeloid leukemic HL60 cells [313,314]. Sb 3+ -induced apoptosis in CCRF-CEM cells increased in the presence of both buthionine sulfoximine (a gamma-glutamylcysteine synthetase inhibitor) and sodium ascorbate, which reduce intracellular glutathione levels in human myeloid leukemic HL60 cells [312,314]. The metal-induced ROS production increase and cell viability decrease showed the order of As 3+ > As 5+ > Sb 3+ > Sb 5+ , and these effects were the main cause of Sb cytotoxicity in these cells [311].
As(III), similar to the other heavy metals above, induced oxidative stress (Figure 1) accompanied by a decrease in CI and CII activities, MPTP opening, an ROS production increase, and an ATP production and ∆Ψ mito decline. However, As 3+ (unlike the heavy metals discussed above) can be classified as a mild thiol effector due to its weak influence on mitochondrial GSH content. As(III) similar to Pb 2+ increases cytoplasm [Ca 2+ ] mobilizing Ca 2+ from the ER, which is followed by mitochondrial dysfunction due to the calcium overload of those organelles. That elimination of the As(III)-induced oxidative stress and apoptosis consequences in the presence of NAC, ascorbate, and selenite is probably due to the significant reversibility of these As(III)-induced processes. It should be noted that this circumstance is indicated by preventing As 3+ -induced apoptosis due to the acceleration of the processes of glutathione biosynthesis or the action of metallothioneins or methyl donors on cells. A few studies on Sb(III) compounds found effects similar to those of As(III). Undoubtedly, the moderate binding of As(III) to the thiol groups of various proteins and enzyme complexes has become the reason for the recent rapid growth in research on the search for and synthesis of arsenic-containing drugs that selectively suppress the growth of cancer cells for the treatment of oncological diseases.
Cr(VI). Mitochondrial research. Cr 2 O 7 2− induced a state 3 and 3U FCCP respiration decrease, an ROS production increase, and ∆Ψ mito decline in isolated RLM energized by CI and CII substrates [315,316]. However, state 4 respiration increased and peaked at 250 µM K 2 Cr 2 O 7 , which was followed by a sharp decrease in respiration [316]. Dichromate weakly affected the inner membrane passive proton permeability; however, the FCCPinduced permeability of the IMM was partially reduced by Cr 2 O 7 2− [316]. RLM took up Cr(VI), which reduced further to Cr(V) in the matrix [317]. Glutathione eliminated these effects of Cr 2 O 7 2− [81,315]. Cr 2 O 7 2− induced the state 3 inhibition and ∆Ψ mito decline in mitochondria isolated from L02 hepatocytes [318].
Cr(VI) compounds are of industrial origin and very rarely occur in nature in the form of crocoite mineralade, PbCrO 4 . Being the most potent oxidizing agent, Cr 2 O 7 2− induced oxidative stress (Figure 1) in addition to lysosomal membrane rupture, GSH oxidation, CI and CII inhibition, a decline in ∆Ψ mito and ATP production, MPTP opening, ROS production, and an increase in lipid peroxidation in experiments with various cells in vitro. At the same time, under acting Cr(VI) compounds, as in the case of the heavy metals considered above, apoptosis ( Figure 2) was induced due to caspase-3 activation with a preceding p53 signal and mitophagy. Antioxidants and ROS scavengers prevented these cytotoxic effects. Cr(VI) toxicity may increase due to the activation of nitrosylation processes in cells, which was followed by the activation of apoptosis by a mechanism involving S-nitrosylation and nitric oxide-dependent stabilization of the Bcl-2 protein [341]. Therefore, cell damage caused by Cr(VI) compounds can provoke carcinogenesis, leading to oncological diseases, including lung cancer, due to the malignant transformation of human lung epithelial cells.
U(VI). Mitochondrial and cell research. UO 2 2+ attenuated mitochondrial respiration in states 3 and 3U DNP and induced both a H 2 O 2 formation increase and a slight calcium retention increase in succinate-energized RLM [342]. UO 2 2+ induced an ROS production and lipid peroxidation increase as well as a reduced glutathione content and ∆Ψ mito decrease in the isolated kidney mitochondria of rats injected with uranyl acetate [343]. The toxicity of UO 2 2+ manifested as CII and CIII inhibition, mitochondrial swelling, ∆Ψ mito decline, and an increase in H 2 O 2 and ROS production and lipid peroxidation as well as a decrease in cell viability, reduced glutathione content, mitochondrial ATP content, the ATP/ADP ratio, cytochrome c release, and outer mitochondrial membrane damage in experiments with primary rat hepatocytes, human dermal fibroblast primary cells, and isolated rat kidney mitochondria (RKM) energized by CI and CII substrates [343][344][345][346]. MPTP inhibitors (carnitine, CsA, and trifluoperazine), antioxidants (catalase), ROS scavengers (mannitol and DMSO), or a U 6+ reduction inhibitor (Ca 2+ ) prevented the UO 2 2+ -induced ∆Ψ mito decrease and lysosomal membrane hepatocyte damage [344]. Ca 2+ blocked the glutathione or cysteine capacity to reduce U 5+ and U 6+ to U 4+ to form O 2 •− in these hepatocytes [344].
Beta-glucan and butylated hydroxyl toluene prevented both these toxic UO 2 2+ effects and the UO 2 2+ -induced outer mitochondrial membrane damage in RKM [347]. Uranyl binding with cyt b 5 , cyt c, and the cyt b 5 -cyt c complex may indicate that UO 2 2+ -induced apoptosis forms a dynamic cyt b 5 -cyt c complex [348]. UO 2 2+ induced apoptosis with the activation of caspases-3, -8, -9, and -10 to indicate a mitochondriadependent signaling pathway in rat kidney NRK-52E proximal cells, rat hepatic BRL cells, HEp-2 cells, and human dermal fibroblast primary cells [342,346,349,350]. There was also UO 2 2+ -induced oxidative stress accompanied by a cell viability decrease, an ROS production increase, a lipid peroxidation increase, a reduced glutathione content decrease, and ∆Ψ mito collapse. Zn 2+ (an effective heavy metal poisoning antidote) protected against UO 2 2+ -induced apoptosis in human kidney HK-2 cells [351]. In addition, Zn 2+ prevented the UO 2 2+ -induced cell viability decrease, cytochrome c release to the cytoplasm, ∆Ψ mito decline, lactate dehydrogenase (LDH) depletion, and increase in ROS production and catalase and glutathione concentrations [351]. UO 2 2+ (similar to heavy metals) initiated apoptotic processes with the activation of caspases-3 and -9 and formed a dynamic cyt b5-cyt c complex. Moreover, uranyl-induced mitochondrial oxidative stress ( Figure 1) manifested as an ROS production and lipid peroxidation increase, a GSH decrease, CII and CIII inhibition, MPTP opening, and ∆Ψ mito decline. The cytotoxic effects of UO 2 2+ were markedly attenuated in experiments with MPTP inhibitors, antioxidants, and ROS scavengers. High uranyl toxicity was found for some bacteria [352]. In addition, uranyl disrupts cellular calcium metabolism. One can hypothesize that uranyl toxicity (together with natural radioactivity) may manifest itself through disturbances in calcium-dependent metabolic processes, exacerbating metabolic disorders. The toxicity of UO 2 was higher than the toxicity of Al and comparable to the toxicity of Cu, Zn, and Pb, but it was lower than the toxicity of Cd and Ag [353].
Modified heavy metals. Cell research. Reduced glutathione was shown to react directly with Ag + ions [94]. Tl 3+ ions unlike Tl + ones oxidized glutathione [354]. The toxic effects of MeHg were more potent than those of Hg 2+ on zebrafish ZF4 and human gingival fibroblasts [153,163]. The co-exposure of AgNPs, Cd 2+ , and Hg 2+ showed increased toxicity in experiments with human hepatocarcinoma HepG2 cells, with AgNP + Hg 2+ being less toxic than AgNPs + Cd 2+ [355]. Nanoparticles (CdO or Ag) inhibited succinate dehydrogenase in BRL-3A hepatocytes [104]. Nano-carbon black with Pb 2+ particles simulating the atmosphere fine particles formed a special complex that activated apoptotic signaling pathways, impaired ∆Ψ mito , and inhibited the lysosomal function in rat alveolar macrophages [356]. Pb 4+ as Pb(acetate) 4 increased intracellular human neuroglobin and ROS production in breast cancer MCF-7 cells and induced mitochondrial apoptosis [357]. Trialkyllead compounds similar to Ca 2+ with valinomycin induced an increase in mitochondrial swelling, state 4 respiration, and K + efflux, as well as a decrease in state 3 respiration, in succinate-energized RLM injected in KCl but not in a KNO 3 medium [358][359][360]. Minerals containing heavy metals with a maximum oxidation state (Tl 3+ and Pb 4+ ) do not occur in nature, and their use in research is purely for laboratory use. Ph 3 Sb(V)O in complex with carvacrol was found to reduce in participating glutathione to a toxic form containing a Ph 3 Sb(III) complex that resulted in apoptosis, mitochondrion damage, and mitochondrial membrane permeabilization in human breast adenocarcinoma MCF-7 cells [361]. The AlF 4 − complex being isomorphous to Pi inhibited beef heart mitochondrial F 1 -ATPase, pig kidney Na + , K + -ATPase, plasmalemmal stomach smooth muscle Ca 2+ , and Mg 2+ -ATPase [362][363][364]. Gold(I)-derived complexes synthesized on the basis of Au[P(Ph) 3 ] + Cl − were proven to be effective trypanothione reductase inhibitors and proposed to treat leishmaniasis [365]. These gold complexes induced oxidative stress with an ROS production increase, mitochondrial damage, and a mitochondrial respiration decrease in experiments with human monocyte-derived macrophage THP-1 cells infected by leishmaniasis parasites.

Conclusions
This review analyzes the causes and consequences of apoptosis and oxidative stress that occur in mitochondria and cells exposed to the toxic effects of different valent heavy metals (Ag + , Tl + , Hg 2+ , Cd 2+ , Pb 2+ , Al 3+ , Ga 3+ , In 3+ , As 3+ , Sb 3+ , Cr 6+ , and U 6+ ). Experiments with different cells and mitochondria showed that the heavy metals under review induced apoptosis characterized by caspase-3 and -9 activation, Bax and Bcl-2 expression, and mitogen-activated protein kinases (ERK, JNK, p53, and p38). Reduced cell viability and oxidative stress were observed, and they manifested as MPTP opening, mitochondrial swelling, an ROS and H 2 O 2 production increase, lipid peroxidation, cytochrome c release, and a reduced glutathione and oxygen consumption decrease as well as cytoplasm and matrix calcium overload due to Ca 2+ release from ER. There was also an ATP synthesis decrease and ∆Ψ mito decline due to CI-CIII dysfunction found in experiments in vitro with both cells and mitochondria. This dysfunction is due to the interaction of some metals (Ag + , Hg 2+ , Cd 2+ , Pb 2+ , As 3+ , and Sb 3+ ) with high-affinity thiol groups of the respiratory complexes and adenine nucleotide translocase. On the one hand, other metals (Al 3+ , Ga 3+ , In 3+ , Cr 6+ , and U 6+ ) induce H 2 O 2 and ROS production as well as lipid peroxidation that results in the function oxidative damage of these respiratory complexes. Some metals (Pb 2+ , Al 3+ , and Ga 3+ ) disturb Fe 2+ metabolism and distort the structure of the iron-sulfur centers of the mitochondrial respiratory chain. At the same time, it should be noted that Tl + among the metals under review possesses significant differences. On the one hand, the toxic effects of Tl + and other heavy metals showed an obvious similarity in experiments with cells. On the other hand, Tl + in experiments with isolated mitochondria did not induce MPTP opening and showed negligible reactions with mitochondrial thiol groups and no inhibition of respiratory enzymes. The toxic effects of Tl + were similar to those of heavy metals only in experiments with calcium-loaded mitochondria. Thus, the similarity of the effects of Tl + and other heavy metals may be due to the increased cytoplasmic calcium concentration induced by these metals. However, the toxicity of thallium being greater than the toxicity of mercury, lead, cadmium, copper, and zinc to humans may be due to the fact that the existing metallothionein-dependent mechanisms do not reduce thallium toxicity, which is in contrast to the toxicity of these heavy metals. Another reason for the thallium toxicity may be our hypothesized decrease in reduced glutathione in the matrix as a result of the reversible oxidation of Tl + to Tl 3+ near the centers of the generation of ROS in the respiratory chain.

Conflicts of Interest:
The author declares no conflict of interest. An analog of ruthenium red SOD Superoxide dismutase tBHP