J. Pineal Res. 2014

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Doi:10.1111/jpi.12132

Journal of Pineal Research

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

REVIEW ARTICLE

A review of metal-catalyzed molecular damage: protection by melatonin Abstract: Metal exposure is associated with several toxic effects; herein, we review the toxicity mechanisms of cadmium, mercury, arsenic, lead, aluminum, chromium, iron, copper, nickel, cobalt, vanadium, and molybdenum as these processes relate to free radical generation. Free radicals can be generated in cells due to a wide variety of exogenous and endogenous processes, causing modifications in DNA bases, enhancing lipid peroxidation, and altering calcium and sulfhydryl homeostasis. Melatonin, an ubiquitous and pleiotropic molecule, exerts efficient protection against oxidative stress and ameliorates oxidative/nitrosative damage by a variety of mechanisms. Also, melatonin has a chelating property which may contribute in reducing metal-induced toxicity as we postulate here. The aim of this review was to highlight the protective role of melatonin in counteracting metal-induced free radical generation. Understanding the physicochemical insights of melatonin related to the free radical scavenging activity and the stimulation of antioxidative enzymes is of critical importance for the development of novel therapeutic strategies against the toxic action of these metals.

Alejandro Romero1, Eva Ramos1,
bal de Los R
ıos2,3, Javier Cristo Egea2, Javier del Pino1 and Russel J. Reiter4 Departamento de Toxicolog
ıa y Farmacolog
ıa, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain; 2
filo Hernando y Departamento de Instituto Teo
utica, Facultad de Farmacolog
ıa y Terape
noma de Madrid, Medicina, Universidad Auto
n Madrid, Spain; 3Instituto de Investigacio Sanitaria, Servicio de Farmacolog
ıa Cl
ınica, Hospital Universitario de la Princesa, Madrid, Spain; 4Department of Cellular and Structural Biology, University of Texas Health Science, Center at San Antonio, San Antonio, TX, USA 1

Key words: chelating properties, free radicals, melatonin, metals, oxidative stress Address reprint requests to Alejandro Romero, Departamento de Toxicolog
ıa y Farmacolog
ıa, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda, Puerta de Hierro s/n 28040-Madrid, Spain. E-mail: [email protected] Received February 17, 2014; Accepted March 11, 2014.

Introduction Metals are crucial for a wide variety of biologic processes of living systems; however, numerous studies have provided evidence that xenobiotic metals, with no physiological functions such as aluminum, cadmium, lead, mercury, and arsenic, can interact with biologic macromolecules causing oxidative damage. Although the molecular mechanisms are not completely understood, the capacity of metals to generate reactive oxygen (ROS) and nitrogen species (RNS), and, thus, to disrupt the maintenance of redox homeostasis is considered the most important event involved in metal-induced toxicity [1]. The metal-induced ROS/RNS generation is directly involved in the induction of epigenetic changes, abnormal cell signaling and uncontrolled cell growth, initiation of cellular injury and the stimulation of inflammatory processes; each of these is pivotal mechanisms that can lead to cancer development [2, 3]. Interestingly, antioxidant therapy protects against metal toxicity by trapping free radicals and maintaining redox homeostasis. In line with this, melatonin is known to be a versatile and ubiquitously functioning molecule [4–11]. The antioxidant abilities of this indoleamine derive from both its direct scavenging of free radicals [12–17] and by increasing the activity and

expression of antioxidant enzymes [18–20]. Conversely, deficiencies in melatonin production or melatonin receptor expression and decreases in melatonin levels likely contribute to numerous dysfunctions and diseases. Thus, its loss is associated with a multitude of pathophysiological changes [21–27]. Considering its diverse actions and given melatonin’s high lipophilicity and the ease with which it crosses morphophysiological barriers, its use as a combination therapy with a wide variety of currently used metals suggests interesting therapeutic perspectives. The purpose of this review is to provide a detailed overview of the current state of knowledge related to the role of metals in the generation of ROS/RNS and tissue injury and to summarize the research performed in recent years on the protective role of melatonin against metal-induced toxicity. Furthermore, we included, from a chemical point of view, a hypothetical representation on the chelating properties of melatonin. Cadmium and melatonin The heavy and nonessential divalent metal cadmium (Cd2+) is the main toxic form of Cd that induces oxidative stress. Although Cd2+ is not a Fenton metal, there are several mechanisms by which Cd2+ indirectly can generate 1

Romero et al. ROS [28, 29]. Human exposure to Cd2+ comes from a variety of sources: drinking water, food, air, and high concentrations are found in cigarettes. Therefore, Cd2+ is an ubiquitous element present in the environment and it is classified as a Class 1 human carcinogen [30]. In an interestingly recent review by Th
evenod and Lee [29], the relevant signaling pathways and action mechanisms that are targeted by Cd2+ as well as involvement of ROS signals are analyzed. In this complex scenario, it should be realized that there are different mechanisms of acute and chronic Cd2+ toxicity. It has been suggested that the mechanisms of acute Cd2+ toxicity elicit a persistent rise in ROS and Ca2+ which disrupt cell function and trigger cell death [31] in both in vivo and in vitro models. Moreover, Cd2+ inhibits antioxidative enzyme activity probably by directly binding to these proteins [32–34], causes depletion of glutathione (GSH) and protein-bound sulfhydryl groups, induces lipid peroxidation, and mediates DNA damage. Conversely, chronic low Cd2+ exposure conditions are more complex, and the roles of ROS are variable depending on experimental conditions.

It is known that ROS/RNS play a pivotal role as signaling molecules in a manner similar to other second messengers. These toxic molecules are induced by physiological or external stimuli including cytokines or metals. Also ROS/ RNS can be generated through nonenzymatic processes that are not highly regulated; they can be also produced by the activity of NADPH oxidases (NOX) which act as signaling molecules [35]. Taking into account the disruption caused by Cd2+ in numerous systems, it would be expected that melatonin would be a therapeutic multipotent agent against damage induced by this heavy metal. At the cellular level, herein, we summarize the main toxic effects and signaling cascades induced by Cd2+ and the protective role of melatonin (Fig. 1). To our knowledge, the first report that documented the protective action of melatonin against Cd2+ was conducted by Kim et al. [36] who demonstrate, in an in vivo model, that melatonin (10 mg/kg b.w., i.p.) co-administered daily with cadmium chloride (CdCl2) restored the reduction in hepatic GSH levels and ameliorated histopathological changes after Cd2+ exposure. A few years

Fig. 1. Model of cadmium pathways inducing cellular stress and the protective role of melatonin. Cd induces endoplasmic reticulum (ER) stress by calcium overload through direct activation of ceramide and binding to protein G-coupled receptors. The increased [Ca2+]c induces calpain–caspase-3-mediated apoptosis. Cd activates directly or indirectly mitochondrial reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation and release the pro-apoptotic regulator cytochrome c (Cyt c) promoting caspase-3-mediated apoptosis. Cd down-regulates Ube2d genes inhibiting the P53 degradation which causes P53 overload inducing apoptosis. Melatonin counteracts Cd damage by blocking caspase-3 and reducing the ER stress caused by ROS/RNS. Melatonin also activates antioxidative enzymes and the GSH/GSSG cycle.

2

Melatonin for protection against metals later, Karbownik et al. [37] showed that lipid peroxidation induced by CdCl2 (1 mg/kg b.w., i.p.) in hamster tissues was prevented differentially by melatonin. It was presumed on the basis of these studies that melatonin reduced cadmium toxicity firstly by acting as a direct scavenger of free radicals which initiated lipid peroxidation processes and secondly by stimulating the activity of antioxidative enzymes. Thus, the pretreatment with three natural antioxidants, curcumin, resveratrol, and melatonin, in all cases, protected against Cd2+-induced lipid peroxidation and ameliorated oxidative damage in Cd2+-treated mice [38]. In addition to direct scavenging and/or the indirect antioxidative properties of melatonin, in an in vivo study, this indoleamine also reduced kidney Cd2+ accumulation [39]. This fact could be due to (i) melatonin’s lipophilic character which allows it to cross cellular membranes permitting the removal of Cd2+, (ii) establishing stable complexes with Cd2+ [40] (see hypothesis postulated herein, Fig. 2), or (iii) inhibition of intestinal absorption of Cd2+. Furthermore, melatonin administration alone or in combination with selenium and vitamin E prevents oxidative stress induced by Cd2+ in the plasma [41], testes [42], liver, and kidney of rats [43]. Knowing that Cd2+ also acts as neurotoxic agent and endocrine disruptor, previous reports have shown the protective effect of melatonin (0.4 and 3 lg/mL in the drinking water) against oxidative stress induced by very low dose of CdCl2 (5 ppm in the drinking water). In these studies, Cd2+ acted on stress marker gene expression including inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) among others, both at the hypothalamic and pituitary level [44–47]. Metallothioneins (MTs) are an important family of proteins endowed with a high capacity to bind heavy metals in biologic systems, thereby protecting against oxidative injury [48]. In an in vitro study on three cell lines, AlonsoGonz
alez et al. [49] revealed that melatonin increased Cd2+-induced gene expression of MT isoform MT-2A in all cell types studied. This isoform is widely expressed in humans, and its induction by melatonin aids in reducing oxidative damage induced by Cd2+. Cellular and molecular mechanisms in Cd2+-induced nephrotoxicity have been widely studied. However, further research is necessary to identify an appropriate therapeutic approach to Cd2+-induced renal injury. Melatonin has shown a substantial ability in preventing oxidative stress and tissue damage resulting from Cd2+ toxicity [50]. The same research group exposed rats in vivo to Cd2+ (5 mg/kg b.w. s.c.) for 22 days and recorded an increase in malondialdehyde (MDA) levels and a reduction in both

Fig. 2. Hypothetical g6-coordination of metals to melatonin through the p-electron density of the benzene-fused ring in a tetrahedral-like fashion.

the activity of superoxide dismutase (SOD) and GSH concentrations in the liver. In contrast, when melatonin was administered with Cd2+, MDA levels and the low enzymatic values found after Cd2+ treatment were restored to control levels [51]. Other important targets for Cd2+ are the testes and cardiovascular system. A recent report confirmed necrosis of seminiferous tubules in Cd2+-treated mice. In this model, melatonin treatment not only alleviated Cd2+-induced histopathological injury but also attenuated testicular HO-1 up-regulation and protected against apoptosis in the testes [52]. Similarly, Cd2+-exposed rats experienced a significant decrease in myocardial antioxidative enzymes; however, simultaneous administration of melatonin and alpha-lipoic acid provided cardioprotection, minimized free radical generation, and maintained the antioxidant status [53]. Based on these findings, it is likely that the administration of exogenous melatonin may be an effective means to prevent Cd2+ toxicity. Presumably, the effective dose required to suppress Cd2+ toxicity would vary according to the amount of Cd2+ to which and individual was exposed. Mercury and melatonin Mercury is a widespread environmental and industrial pollutant; consequently, it is practically impossible for humans to avoid exposure to some of the forms of mercury. It is recognized as one of the most dangerous environmental contaminants. Humans and animals are exposed to numerous chemical forms of mercury, including elemental mercury vapor (Hg), inorganic mercurous [Hg(I)], mercuric [Hg(II)] and organic mercuric compounds [28, 54], all of which induce severe alterations in tissues of both animals and man. Also, it functions as an endocrine disruptor [55–57], in immunotoxicity [55], reproductive toxicity [58–60], neurotoxicity [61–64], cardiovascular toxicity [65], nephrotoxicity [66, 67], genotoxicity [68, 69], gastrointestinal toxicity with ulceration and hemorrhage [70–72] and it also influences organismal survival [73]. Mercury also crosses the placental barrier [74] and the blood–brain barrier and accumulates in different brain areas including the cerebellum and cerebral cortex [75, 76]. This became obvious in the tragic epidemics in Japan (Minamata disease) and in Iraq [77]. There is no single specific mechanism of toxicity ascribed to mercury, but it likely involves multiple coordinated effects on several parallel processes in the cell. Mercuric ions have a high affinity to bind to reduced sulfur atoms, especially those on endogenous thiol-containing molecules, such as GSH, cysteine, MTs, homocysteine, N-acetylcysteine (NAC), and albumin [28, 78]. It has been proposed that a mechanism of toxicity of mercury may be via binding to thiol groups, thereby damaging DNA, RNA, membrane structure, and proteins [28]. Moreover, an imbalance in the antioxidant protective mechanisms leading to oxidative stress in cells has been identified as a common factor in mercury exposure [79]. Thus, it was reported that treatment with mercury induces a dramatic rise in ROS generation leading to lipid peroxidation, protein degradation, and finally to cell death [72, 80, 81]. 3

Romero et al. Elimination of free radicals by treatment with antioxidants and or free radical scavengers leads to a reduction in the toxicity induced by mercury exposure [82, 83]. Numerous reports have documented the protective actions of melatonin in various models of oxidative stress due to its high efficacy as a free radical scavenger and indirect antioxidant [84–90]. Melatonin, as a direct free radical scavenger and indirect antioxidant, can detoxify numerous ROS including hydrogen peroxide (H2O2), hydroxyl radical (
OH), peroxyl radicals (ROO
), and singlet oxygen (1 O2 ), and also RNS including nitric oxide radical (NO
) and peroxynitrite (ONOO) [91, 92]. Moreover; it stimulates the activities of enzymes that metabolize reactive species, thus preserving the structural and functional make up of subcellular organelles [69]. Several reports using in vivo and in vitro models showed that mercury causes genotoxicity with a delay in the cell cycle, changes in the normal pattern of chromosomal distribution during cellular division, and causes structural chromosomal aberrations which lead to the failure of DNA repair processes [68, 69, 93–95]. Mercury genotoxic is believed to be attributable to its pro-oxidative effects which damage purine–pyrimidine bases and the deoxyribose skeleton in the DNA leading to reduced cell cycle kinetics [69, 93]. These changes in cell cycle kinetics and chromosomal anomalies are prevented by supplementation with antioxidants. Melatonin has been useful in reducing the toxic effects associated with certain classes of chemotherapeutic agents, mutagens, and carcinogens, acting both as an indirect antioxidant and radical scavenger [96]. Moreover, Purohit and Rao [69] reported that melatonin or a-tocopherol alone reduces significantly the genotoxic actions induced by mercury and when given in combination, a much better amelioration is observed. Kim et al. [73] reported that 6-week-old mice treated with methyl mercury (40 mg/g) had a marked reduction in survival rate over the period 30–35 days of treatment, and when this toxic metal was co-administered with melatonin (20 mg/ mL), 100% of the mice survived, likely related to the potent antioxidative action of the indoleamine. Mercury accumulates in erythrocytes and cardiac tissues, causing detrimental effects on the cardiovascular system including a reduction in arterial blood pressure [97, 98], systemic and pulmonary vasoconstriction [99], lowering of myocardial contractility, and heart failure [100, 101]. The mechanisms behind mercury’s toxicity in the cardiovascular system are not completely understood. Cardiotoxicity induced by inorganic and organic mercury seems to be a result of impairment of hemodynamic parameters, loss of baroreceptor control, and elevated oxidative damage [65]. Detoxification of free radicals by treatment with antioxidants and or free radical scavengers has been shown to greatly reduce myocardial dysfunction induced by mercury exposure [82]. Thus, melatonin was shown to have cardioprotective potential in ameliorating mercuryinduced injury via (i) its metal-chelating activity, (ii) reducing mercury accumulation, (iii) repairing neural regulatory mechanisms of arterial blood pressure control, or (iv) increasing antioxidant enzyme activity and reducing free radical-induced cytotoxicity to myocardiocytes and endothelial cell [65]. 4

Many studies have shown that mercury exposure induces neurotoxicity due to dyshomeostasis of neurotransmitters, cytoarchitectural alterations in neural organization, increased oxidative stress, and elevated cell death [61–64]. Rao et al. [102] evaluated melatonin’s protective action induced by mercuric chloride in the cerebral and cerebellar cortices and in the brainstem. Mercury elicited the depletion of enzymatic activities such as adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase, alkaline phosphatase, acid phosphatase and altered glycogen, total protein, and lipid peroxidation levels in these brain areas, thereby affecting their respective functions. Co-treatment with melatonin (5 mg/kg b.w. i.p.) protected against the neural changes induced by mercury. Later, Rao and Purohit [103] documented fine structural alterations including a discontinuous myelin sheath around axons, swollen mitochondria, disfigured nuclei as well as nuclear membrane changes after the treatment of mercuric chloride; melatonin effectively reduced the enzyme and structural alterations exerted by mercury intoxication. It has also been suggested that mercury may be involved in some neurodegenerative disorders such as Alzheimer disease (AD). Hence, it was reported that mercuric chloride induces oxidative stress, amyloid-beta peptide (Ab) production, and elevated phosphorylated tau levels in neuroblastoma SH-SY5Y cells. These effects are reduced and/or totally reversed by treating the cells with melatonin. Thus, the indoleamine greatly attenuated mercury-induced oxidative stress, Ab fibrillogenesis and release, and tau hyperphosphorylation [104]. Mercury compounds also are known to affect testicular spermatogenic and steroidogenic functions in experimental animals and man [105]. Suppression of sperm motility by mercury has been reported in different mammalian species including human in association with decrements in sperm count, and altered sperm metabolism and morphology [59, 60, 93]. Mercury affects accessory sex gland function in rats and mice by producing an androgen deficiency [59]. ROS are important mediators of normal sperm function and are involved in the induction and development of sperm hyperactivation, capacitation, and the acrosome reaction [106]. But excessive production of ROS above normal levels, as mercury is capable of doing, results in lipid peroxidation and membrane damage leading to loss of sperm motility [107], inactivation of glycolytic enzymes [108], damage to the acrosomal membranes [108], and DNA oxidation, which render the sperm cell unable to fertilize the oocyte, or produce a viable pregnancy [109]. The inhibition of steroidogenesis and spermatogenesis by mercury is related to the induction of oxidative stress, lipid peroxidation, and decreasing antioxidant parameters. Melatonin when co-administered with mercury reduces oxidative stress, lipid peroxidation and recovers the androgenic production and testicular spermatogenic functions [105]. Mercury is a known endocrine disruptor, and the most often affected hormones include thyroxin, insulin, estrogen, testosterone, and adrenaline [110]. These agents are responsible for the maintenance of homeostasis, reproduction, development, and behavior [110]. When exposure occurs, mercury levels significantly increase in thyroid of animals and humans [105, 111, 112]. Mercury blocks

Melatonin for protection against metals thyroid hormone production by occupying iodine-binding sites and inhibiting the action of this hormone [105]. Mercury causes hypothyroidism, damage of thyroid RNA, autoimmune thyroiditis, and impairment of conversion of thyroxine to its active form triiodothyronine [105, 113]. Chronic intake of mercury for more than 90 days results in signs of mercury poisoning, together with decreased uptake of iodine and depression of thyroid secretion [57, 114]. Rao et al. [102] reported the mercuric chloride exposure in rats decreased thyroid activities of ATPase and succinate dehydrogenase (SDH) and reduced the generation of ATP, revealing alterations in the oxidative energy metabolism as a result of lesions in tricarboxylic acid cycle by binding with sulfhydryl groups of proteins. They also documented the existence of oxidative stress induced by inorganic mercury in the thyroid, which imposed a significant decline in levels of SOD, catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GRd), and GSH followed by elevated level of lipid peroxidation and an inhibition of thyroid function. Melatonin co-administration with mercuric chloride stimulated the antioxidative enzymes, CAT and GPx, and caused partial recovery in SDH and ATPase activities. Sener et al. [81] observed mercuric chloride toxicity at the level of the kidney, liver, and lungs; this damage was observed to be associated with increases in lipid peroxides, indicating oxidative tissue damage, as well as a rise in myeloperoxidase activity due to neutrophil infiltration, and also a significant reduction in GSH levels. Treatment with antioxidants such as melatonin protected against acute mercuric chloride toxicity by reducing of free radical production and preventing neutrophil infiltration, as well as promoting GSH synthesis. Administration of the melatonin both before and after mercuric chloride administration did not seem to afford additional protection in comparison with a single administration subsequent to mercuric chloride treatment. Mercury is an established nephrotoxicant in animals and humans where it affects the pars recta (S3 segment) of the proximal tubules [28, 66, 115]. Tubular necrosis and kidney damage, induced both in vitro and in vivo, is attributed to oxidative damage [116, 117]. As shown immunohistochemically, in the rat kidney mercuric chloride induces specific stress proteins related to mitochondrial abnormalities [118]. Stress proteins are universally conserved proteins that are a reliable index of repair in injured renal cells [67]. Melatonin reduces nephrotoxicity if administered either before or after the mercury [81, 119]. Its antioxidant effect is corroborated by reduced expression of a mitochondrial chaperone, GRP75, MT, and iNOS in rat proximal convoluted tubules [66]. In addition, maintenance of regular morphology and mitochondrial size and density in S3 segments by melatonin were shown using ultrastructure and morphometric analyses. Arsenic and melatonin The metalloid arsenic (As) is a natural environmental contaminant and a well-documented carcinogen [120] that exists mainly in two biologic oxidation states, arsenite [As (III)] and arsenate [As (V)]. The most toxicologically

potent As compounds are the trivalent forms [121] that can react with the sulfhydryl groups of proteins to inhibit many biochemical pathways. Pentavalent forms, which are less toxic, are phosphate analogues that uncouple oxidative phosphorylation. However, humans are exposed to both trivalent and pentavalent forms. Inorganic As can be either methylated to form monomethylarsonic acid [MMA (V)] or dimethylated As in dimethylarsinic acid [DMA(V)]. Complex metabolism and As biotransformation may play a pivotal role in the toxic and carcinogenic effects. For more than one decade, it has been known that methylation of inorganic As was considered the detoxification mechanism [122]. However, recent reports have shown the existence of trivalent intermediates [123], monomethylarsonous acid [MMA(III)], and dimethylarsinous acid [DMA(III)], in human urine which are more reactive and carcinogenic than pentavalent compounds. Thus, methylation of As would not be a detoxification mechanism. Currently, it has been established that As metabolism may follow two pathways, on the one hand, reduction and oxidative methylation, and on the other hand, GSH conjugation that plays a key role in both the enzymatic and nonenzymatic reduction in pentavalent arsenicals to the trivalent state (Fig. 3). Considering that As is related to diverse pathologies such as hepatic and renal disorders, cardiovascular dysfunction, neurological defects and that it has carcinogenic actions, the exact action mechanism of arsenicals is rather complicate and poorly understood. In recent years, oxidative stress has gathered strength as a likely mechanism of As toxicity [124]. During As exposure, the production of ROS alters the homeostatic balance of antioxidant defenses triggering oxidative stress [125]. Several reports have documented generation of ROS/RNS during arsenic metabolism [126, 127]. Thus, GSH depletion due the consumption induced by As metabolism may be an important step in initiating and spread of free radicals, which are likely to play an important role in the early stages of carcinogenesis. ROS generation induced by As involves pro1 duction of the superoxide anion (O
 O2 ,
OH, 2 ),
hydrogen peroxide (H2O2), ROO , and also RNS such as the NO
. Furthermore, changes in antioxidant enzymes such as CAT and SOD were observed in in vitro models after As exposure [128, 129]. Reactive oxygen species are critical elements in the signal transduction pathways and transcription factor regulation. Several studies have shown that exposure to As and/ or As-induced ROS generation activate a cellular transcription factors including NF-jB, AP-1, and p53 and signaling pathways through MAP kinases [130, 131] (Fig. 4). Low doses of As increase the activity of the antioxidant enzymes including SOD, CAT, GPx, glutathione S-transferase (GST), and GRd; however, chronic exposure to this metal results in reductions in these enzyme activities. It also acts as modulator of the activity of thioredoxin reductase, heme oxygenase reductase, and NADPH oxidase [132]. Although not directly mutagenic, As is considered a comutagenic and genotoxic metal, as observed in in vitro and in laboratory animals and humans. It induces deletion mutations, oxidative DNA damage, DNA strand breaks, 5

Romero et al.

Fig. 3. Schematic diagram of arsenic metabolism.

sister chromatid exchanges, aneuploidy, transforming activity, and genomic instability [133]. Nesnow et al. [134] using a supercoiled /X174 DNAnicking assay, showed that DNA injury activity of DMA (III) is an indirect genotoxic effect mediated by ROS. The use of lM concentrations of tiron, melatonin, or trolox inhibited the DNA-nicking activities of both MMA(III) and DMA(III). However, to cause in vivo oxidative damage, the authors used high concentrations of As to promote cytotoxicity. The co-administration showed that melatonin slightly inhibits arsenite-induced in vitro urinary bladder cytotoxicity, but had no effect on the other arsenicals [135]. Perhaps in these studies, the dose of melatonin used (1000 ppm) and the route of administration chosen (oral) reduced its efficacy. When rats were injected with sodium arsenite (5.55 mg/kg b.w. 6

i.p.) for a period of 30 days, significant reductions in enzyme activities in kidney and liver were observed. Melatonin supplementation (10 mg/kg b.w. i.p.) for the 5 days prior to sacrifice reversed arsenic-induced metabolic toxicity [136]. Using the same doses of melatonin and sodium arsenite, these authors also observed changes in the antioxidant system after As exposure including reductions in the activities of SOD and CAT and suppression of the level of GSH and GRd activity in liver and kidney. Melatonin supplementation completely restored all parameters altered by As to control levels with exception of CAT [137]. Oxidative stress, after the application of sodium arsenite, also was evaluated in the nigrostriatal dopaminergic system of rat brain by Lin et al. [138]. This report demonstrated that the neuroprotective effects of melatonin

Melatonin for protection against metals

Fig 4. Arsenic-induced tumorigenesis and differentiation through cell signaling epidermal growth factor receptor (EGFR) via MAP kinases. Reactive oxygen species (ROS) generated after exposure to arsenic also activates molecules such as Ras and ERK and IjB complex. IjB complex phosphorylation leads to p65 and p50 migration to the nucleus which results in activation of pro- and anti-inflammatory cytokines which play important roles in carcinogenesis. ROS also activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a key transcription factor that regulates the cellular antioxidant response. The oxidation of As3+ to As5+ under physiological conditions results in the formation of H2O2. Melatonin counteracts the toxic and tumorigenic effects caused by As, by (i) scavenging ROS production and stimulating antioxidative enzymes, (ii) increasing GSH levels and, (iii) modulating transcription factors such as NFjB and Nrf2.

against As-induced apoptosis involved both mitochondrial and endoplasmic reticulum processes. These data suggest that melatonin may be a good therapeutic tool to reduce oxidative stress caused by As in the central nervous system (CNS). A subsequent report evaluated the role of Asinduced oxidative stress in peripheral neurotoxicity using dorsal root ganglion explants [139]. Taking into account the pivotal role that the mitochondrial and endoplasmic reticulum pathways play in oxidative stress, melatonin likely worked via these pathways to inhibit As-induced apoptosis and oxidative stress (Fig. 4). When compared with two antioxidative thiols, GSH and NAC, melatonin was more neuroprotective than were these agents. These two studies demonstrated the neuroprotective efficacy of melatonin against As in both the central and peripheral nervous systems. Arsenic also induces apoptosis and oxidative stress in rat testes. Administration of sodium arsenite (NaAsO2) (5 mg/kg/day, intragastrically) increased the number of apoptotic germ cells and the biomarker of lipid peroxidation, MDA, while reducing SOD, CAT, and GPx activities. The concurrent treatment of rats with NaAsO2 plus

melatonin (25 mg/kg/day, i.p.) counteracted As-induced testicular apoptosis and oxidative stress [140]. The abundance of polyunsaturated fatty acids (PUFA) in the brain renders it particularly vulnerable to ROS. The role of lipid peroxidation as a major factor in Asinduced oxidative stress is well known. In an in vivo study, melatonin inhibited arsenite-mediated lipid breakdown in a concentration-dependent manner in rat brain [141]. The potential of melatonin as an antigenotoxic agent against As was evaluated by Pant and Rao [142] using the comet assay to evaluate As-induced DNA damage. They reported that melatonin protected human blood cells from the exposure of pro-oxidant actions of As. The exact molecular mechanisms of As toxicity and carcinogenesis have yet to be fully identified. Current views, in addition to espousing increased oxidative stress, also advance genetic changes and altered gene expression. Some evidence suggests that inflammation may also have a role in the As-mediated toxicity. Wang et al. [131] demonstrated that cyclooxygenase-2 (COX-2) can be a target of As-mediated toxicity in human uroepithelial cells. In 7

Romero et al.

Fig. 5. Model of cellular iron and copper homeostasis and possible interaction with melatonin (Mel). Fe3+ iron-loaded transferrin (Tf) binds to TR receptor (TfR); this complex is internalized into the endosome, where Fe3+ is reduced to Fe2+. Fe2+ is transported to the cytoplasm. The labile iron pool is defined as weakly bound iron, on average, in the +2 state. Ferritin is the iron high-capacity storage protein. Ceruloplasmin (CP) transports plasma Cu+; Cu+ enters from the blood through copper transport proteins (CTR) to the cytosol. Inside the cell, it is distributed by specific chaperones to the target protein. Copper but not iron has an effective excretion mechanism. Through the Fenton reaction these metals transform H2O2 into •OH, one of the most toxic reactive oxygen species (ROS) in nature. The imbalance of iron or copper homeostasis can increase Fe2+ or Cu+ levels, and in this way induce oxidative stress. Melatonin is a free radical scavenger, antioxidant enzyme activator and as iron chelator, inhibiting metal-mediated oxidative injury.

this study, NaAsO2 increased the COX-2 expression and activity through several modulators of the MAPK pathway (P38 and JNK) involved in cellular differentiation, inflammation, and apoptosis. Melatonin treatment (0.5 mM) reduced the generation of intracellular ROS and the expression of COX-2 mRNA induced by NaAsO2. The same research group found that low dose arsenic induced a significant rise in ATF2 (activating transcription factor and a member of AP-1) expression, which plays an essential role in the cellular stress response. Melatonin treatment and JNK or p38 inhibitors decreased significantly arsenic-induced ATF2 expression [130]. Lead and melatonin Lead is an environmental and occupational toxicant which is known to damage vital organs and suppress cellular processes [143, 144]. The main negative effects of lead are neurotoxic, genotoxic, and hematological damage [145]. Lead neurotoxicity results in behavioral changes and neurochemical alterations in neurons as a result of perturbations and disruption of main structural components of the 8

blood–brain barrier, through primary injury to astrocytes and due to secondary damage of the endothelial microvasculature [146]. Lead treatment results in a significant accumulation of this metal in all brain regions with maximal levels occurring in the hippocampus. The best-documented mechanisms of lead genotoxicity are indirect and include inhibition of DNA repair and production of free radicals [147]. Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis [148]. Similar to other persistent toxic metals such as arsenic, cadmium, and mercury, lead damages cellular components at least partially via elevated levels of oxidative stress. The pathogenetic actions of lead are multifactorial as it directly interrupts the activity of enzymes, competitively inhibits absorption of important trace minerals, such as calcium and zinc, and deactivates antioxidant sulfhydryl pools [149]. Free radical-induced damage by lead is accomplished by two independent, although related, mechanisms [125]. The first involves the direct formation of ROS including 1 O2 , hydrogen peroxides, and hydroperoxides, and the second

Melatonin for protection against metals mechanism is achieved via depletion of the cellular antioxidant pool, inhibiting GRd and d-aminolevulinic acid dehydrogenase (ALAD). A direct correlation between blood lead levels, ALAD activity, and erythrocyte concentrations of MDA has been observed among workers exposed to lead. There have been several attempts to use melatonin to ameliorated lead toxicity. El-Sokkary et al. [86] investigated the neuroprotective action of melatonin against lead-induced neurotoxicity in rats. In this work, melatonin almost completely attenuated the lead-induced increase in lipid peroxidation products and restored GSH levels and SOD activity. The metal also caused severe cellular damage and reduced neuronal density in the hippocampus and striatum. Again, melatonin prevented the structural damage and maintained neuronal density. The neuroprotective effect of melatonin in lead treated animals is related to its direct radical scavenging actions and its indirect antioxidant effects [150]. In addition to its antioxidant effects, the authors discuss several other mechanisms that may be involved in the neuroprotection mediated by melatonin; these include interactions with calmodulin, blockade of rises in intracellular Ca2+, changes in gene expression and activities of antioxidant enzymes and improved efficiency of mitochondrial oxidative phosphorylation. In the cultured human neuroblastoma cell line, SH-SY5Y, exposed to low levels of lead, melatonin also restored lead-induced GSH depletion and protected against apoptosis by inhibiting caspase-3 activation [151]. Melatonin prevented oxidative DNA damage in blood lymphocytes likely due to its ability to scavenge ROS [152]. Mart
ınez-Alfaro et al. [153] investigated the effect of melatonin on DNA damage and repair in lymphocytes of rats subchronically exposed to lead. The authors showed that low levels of lead acetate treatment induced oxidative stress, while melatonin administration reduced the toxic lead effects, but its efficacy depends on the concentration of lead to which the cells are exposed. The dose of lead acetate administered correlated with the level of lead in the blood and the extent of DNA damage in lymphocytes. Finally, administration of melatonin in conjunction with lead treatment reduced hepatic and renal toxicity in rats treated with 100 mg/kg lead for 30 days [154]. Melatonin co-treatment significantly inhibited the levels of lipid peroxidation, stimulated SOD activity and GSH concentration, restored the observed morphometric parameters, and prevented the histopathological changes in the liver and kidney. Aluminum and melatonin Aluminum is not an essential element; indeed, to date, no biologic function has been determined for aluminum. However, its presence in an organism constitutes a toxicity risk [155]. Aluminum accumulation in organs or tissues results in molecular damage or dysfunction and local concentrations of this metal usually correlate with these effects. In biologic systems, the aluminum oxidation state is Al3+. It is typically found as insoluble complexes, and therefore, its bioavailability is highly reduced.

Aluminum has long been used in industry, medicine, agriculture, and water treatment, and thus, exposure to this metal is extensive. Although aluminum is ubiquitous, it has a limited bioavailability due to its insolubility; only a small fraction of aluminum present in the diet is absorbed; most aluminum is rapidly eliminated from the body. Occupational exposure to aluminum is widespread; when the particles are inhaled, they are deposited in lungs, released into blood, and thereafter they are distributed to brain were they accumulate and exert neurotoxic effects [156]. The relationship between occupational exposure and neurobehavioral impairments is controversial; some epidemiological studies indicate there is a relationship [157, 158], while others did not find any [159]. Even though aluminum has no redox capacity in biologic systems, a number of data show that aluminum, at high concentrations, causes oxidative stress through multiple mechanisms [160]. Aluminum binds to many biologic macromolecules through interactions that may displace other biologic cations from their binding site [161]. High levels of aluminum in brain are presumably related to a number of neurodegenerative disorders such as dialysis encephalopathy, AD, and Parkinson disease (PD) early 1960s [162]. The fact that oxidative stress is associated with most neurodegenerative disorders together with the high concentrations of aluminum found in brain regions led to the increasing strong support that this metal may be related to the etiopathology of AD [155, 163]. Although aluminum is clearly involved in the etiology of AD, whether it plays a major or a minor role remains unclear [164, 165]. In recent decades, numerous studies report the pro-oxidant actions of aluminum in specific neurological areas as well as the protective role of melatonin. Esparza et al. [166] reported that after intraperitoneal administration of aluminum (5 mg/kg 8 weeks) to rats, melatonin (10 mg/kg/day 8 weeks) reduced aluminum-induced prooxidant effects in several neural areas (cerebral cortex, hippocampus, and cerebellum) compared with rats treated exclusively with aluminum. As described in detail below, findings published in a variety of reports confirmed that aluminum accumulates in several brain regions and alters oxidative stress markers such as GST, GSH, GSSG, SOD, GPx, CAT, and the levels of thiobarbituric acid reactive substances (TBARS), while melatonin displays protective effects against all aspects of aluminum-mediated damage. In aluminum-exposed rats (7 mg/kg/day), oxidative stress markers and gene expression of CuZnSOD, MnSOD, GPx, and CAT, as well as the beneficial role of melatonin (10 mg/kg/day), concurrently administered, were evaluated in hippocampus, cerebral cortex, and cerebellum. The results demonstrated that in aluminum-exposed animals, melatonin promotes the activity of the antioxidant enzymes GST, CAT, and SOD, compared with their activities in control and only aluminum-exposed animals [167, 168]. Moreover, aluminum increased significantly TBARS levels, an index of elevated lipid peroxidation; as in many other studies, melatonin greatly mitigated the degree of lipid decomposition [167, 168]. 9

Romero et al. The protective role of melatonin (10 mg/kg/day in drinking water, 6 months) in the regulation of antioxidant enzyme gene expression (CAT, GRd and SOD) in the hippocampus, cerebral cortex, and cerebellum has been evaluated in aluminum-exposed (1 mg/g of diet for 6 months) rats [169, 170]. Levels of aluminum were elevated in all animals that consumed the metal. Melatonin, when orally administered to these rats, exerted an antioxidant action by increasing mRNA levels of CAT, GRd, and SOD in hippocampus, cerebral cortex, and cerebellum [169, 170]. Unexpectedly, in these investigations, aluminum did not significantly elevated lipid peroxidation [169, 170], possibly due to the lower exposure to aluminum compared with other studies or the rather poor oral bioavailability of aluminum. Abd-Elghaffar et al. [171] carried out a study where rabbits that were orally treated with aluminum (20 mg/L in drinking water) and displayed atrophy and apoptosis of cerebral cortical and hippocampal neurons. The morphological changes, as well as the degree of lipid peroxidation and inhibition of SOD, were minimized by postadministration of melatonin (10 mg/kg b.w., 15 days), and melatonin also markedly ameliorated aluminum-induced neurotoxicity [171]. More recently, in a similar study in mice in which aluminum (3.5 mg/kg b.w., 6 weeks) and melatonin (7 mg/kg b.w., 6 weeks) were administered by intraperitoneal injection, aluminum-induced structural and oxidative damage to the medulla of rats, while melatonin, once again, mitigated this induced damage [172]. Aluminum clearly accumulates in brain regions in which damage becomes apparent in neurodegenerative disorders. Invariably, melatonin minimizes aluminum-induced damage in these regions when oxidative injury is involved. These findings make melatonin worthy of investigation as a possible therapeutic agent for neurodegenerative diseases. Biologic membranes are complex, wherein numerous molecules can be targeted by aluminum; these are phospholipids that are negatively charged, thus making these bilayer lipids a priority binding site for aluminum. Several recent studies in biologic membrane models showed that aluminum causes a dose-dependent rises in in vitro lipid peroxidation [173–176]. This has been studied in human platelet membranes where aluminum induces lipid peroxidation and where melatonin is capable to protecting against aluminum toxicity. In summary of these reports, they show that aluminum dose dependently increases the formation of lipid peroxides and melatonin dose dependently inhibits the formation of the lipid peroxides induced by aluminum [174, 175]. To test melatonin’s protective effect on aluminum-induced lipid peroxidation in rat synaptosomal membranes, Mill
an-Plano et al. [173] incubated AlCl3 (0–1 mM) with FeCl3 (0.1 mM) + ascorbic acid (0.1 mM). Aluminum, as previously reported [174, 177], promoted iron-initiated lipid peroxidation increased levels of both MDA and 4-hydroxyalkenal (4-HDA). Co-incubation with melatonin (0.1, 1 and 5 mM) resulted in a reduction in MDA and 4-HDA levels. Melatonin at 5 mM significantly limited MDA and 4-HDA levels to each concentration of AlCl3 tested [173]. More recently, Albendea et al. [176] confirmed these findings and, in 10

addition, showed that the highest concentration of aluminum (1 mM) tested caused significant protein oxidation, as measured by protein carbonyls. At this aluminum concentration, melatonin (1–10 mM) successfully also lowered protein carbonyl levels. Collectively, the reported results are consistent with melatonin’s protection against aluminum-induced molecular damage to a variety of molecules. The protective role of melatonin in mitochondrial and synaptosomal membranes may be due, in part, to its ability to stabilize membranes against free radicals [178–180] in addition to the benefits due to its antioxidant activities. It is well known that aluminum accumulation in the kidney is associated with alterations in the renal function [181–183]. After chronic exposure in rats, aluminum accumulates in renal tissue causing several changes in renal physiology which probably relate to the associated elevated reduction in GSH, GPx, and CAT [184, 185]. Combined administration of melatonin and aluminum significantly limited TBARS formation, normalized GSH levels and partially reversed GPx and CAT activities. However, altered renal function was only partially restored by melatonin and did not alter the accumulation of aluminum [185]. Melatonin invariably reverses lipid peroxidation [186] and partially restores the loss of CAT activity [186, 187] in aluminum-exposed rats. This metal also causes hepatic dysfunction by decreasing hepatic antioxidant enzyme activities, while pre-administration of melatonin attenuates this aluminum-mediated toxicity [188]. Using adsorptive cathodic stripping voltammetry (AdCSV), Lack et al. [189] showed that melatonin interacts with aluminum. The binding of this metal by melatonin may provide protection against aluminum-induced damage. However, in a number of other investigations, melatonin did not seem to act as an aluminum chelator [168, 169, 190]. Thus, at this stage, any role of melatonin’s protection against aluminum being a result of the ability to bind the metal remains in doubt. Chromium and melatonin Chromium is one of the most common elements in the Earth and exists in several oxidation states. Hexavalent chromium [Cr(VI)] compounds, such as chromate (CrO2 4 ) and dichromate (Cr2 O2 7 ), have long been recognized as potent respiratory toxins. Inhalation of particles containing Cr(VI) (dusts, mists, and fumes) is associated with several respiratory diseases, including lung cancer, chronic bronchitis, pulmonary fibrosis, and asthma [191–194]. Chromate predominates under physiological conditions (pH > 6), and dichromate is the predominant form under acidic conditions. Chromate closely resembles sulfate (SO2 4 ) and enters cells via an anion carrier [195], but Cr(VI) compounds also readily cross the skin [193]. Cr(VI) is not itself the toxic species. Once inside the cell, Cr(VI) is quickly reduced to Cr (III), the next stable oxidation state. Cr(III) species do not easily cross cell membranes because they are generally insoluble. Cr(VI) is reduced through reactive intermediates such as Cr(V) and (IV) to the more stable chromium (III) by cellular reductants including GSH, vitamins C and B2, and flavoenzymes [196, 197]. Moreover, this reductive

Melatonin for protection against metals process also causes the generation of ROS. The Cr (VI)/(V) redox couple serves as a cyclic electron donor in a Fentonlike reaction to produce active oxygen species, resulting in the induction of DNA damage [197, 198]. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of peroxiredoxin (Prx) and thioredoxin (Trx) [199, 200], hence increasing the oxidizing state of cells. Melatonin and its metabolites are highly effective in scavenging the highly toxic
OH [201, 202]. Several studies focused on the ability of melatonin to reduce Cr-induced oxidative DNA damage in vitro. Qi et al. [203] showed that melatonin reduced DNA damage produced by CrCl3 plus H2O2. In this study, melatonin was more effective than vitamin C and E in blocking the genotoxicity of Cr (III). In another report, Qi et al. [204] compared the ability of melatonin to reduce Cr-induced oxidative DNA damage with other indoles related to melatonin; these included pinoline, NAC, 6-hydroxymelatonin, and indole3-propionic acid. The authors showed that the ability of pinoline and melatonin to reduce Cr(III)-mediated 8-OHdG formation was found to be equivalent and more effective than the other indoles tested. In these two works, the authors mention that the protective action of melatonin against Cr(III)-induced DNA damage may be related to its direct
OH scavenging action and due to its ability to lower H2O2 concentrations. Moreover, the authors point to the possibility that melatonin could chelated chromium in the same manner in which it reportedly chelates other heavy metals such as aluminum, cadmium, iron, copper, and lead [40]. L
opez-Burillo et al. [205] observed individual and synergistic actions of melatonin and other antioxidants (vitamin C, a-lipoic acid) on oxidative DNA damage produced by CrCl3 plus H2O2. In combination with melatonin, both vitamin C and a-lipoic acid showed synergistic actions in reducing
OH produced by Cr(III)/H2O2. Susa et al. [206] examined whether melatonin has an antioxidant effect on Cr(VI)-induced DNA damage in primary cultures of rat hepatocytes. Moreover, the authors examined by electron spin resonance (ESR) spectrometry the influence of melatonin on
OH formation induced by Cr(V). In this study, the authors concluded that melatonin caused a decrease in the DNA damage, cytotoxicity, and lipid peroxidation caused by Cr(VI), without affecting Cr uptake and chromium distribution in cells, possibly through its ability to increase cellular levels of vitamins C and E and to directly scavenge the
OH. Iron and melatonin Iron (Fe) is an essential and bioactive element required for cellular metabolism, proliferation, and differentiation. The quantity of iron in the body is tightly associated with the control of its absorption; an overload in tissues may cause serious damage. The average human body level is 4–5 g of iron firmly bound/complexed, about a 65% to hemoglobin, 10% is a constituent of myoglobin, cytochromes, and iron-containing enzymes, and 20–30% is bound to the iron storage proteins, ferritin and hemosiderin. Ferritin is a low-affinity and high-capacity storage protein; it can store up to 4500 atoms of iron per protein macromolecule.

Transferrin is a high-affinity and low-capacity protein that it can only store two atoms of Fe(III) per molecule and this protein transports iron in the plasma. Essential processes including oxygen transport, energy production, and DNA synthesis depend on Fe-containing proteins [207]. Organisms, therefore, possess proteins that preserve iron homeostasis and maintain most of the iron sequestered with only trace amounts of the metal remaining free as nonchelated or loosely chelated iron available for catalyzing free radical reactions [208, 209]. Iron’s capacity to exchange one electron in biologic reactions is extraordinary. When one or more of its six ligand-binding sites are not tightly bound, iron engages in one electron exchange reactions with the potential of producing free radicals. It possesses incompletely filled d-orbitals and has a maximal oxidation state of 6+, but only the 2+ and 3+ states are common in biologic environments. Iron is an intrinsic ROS producer. Fe(II) is unstable and tends to react with molecular oxygen forming Fe(III) and O
 2 . The electronic structure of iron and its capacity to undergo redox cycling reactions by accepting [Fe(III) + 1e ? Fe(II)] or donating [Fe(II)  1e ? Fe(III)] electron(s), places iron as a leading component in the production and metabolism of free radicals in biologic systems. However, this capacity also makes iron an essential component of cytochromes, the oxygen-binding molecules, hemoglobin and myoglobin, and many enzymes. Free radical-mediated oxidative stress in cells is one of the main causes of alterations in cellular structure and function due to iron overload [210]. A known and common source of free radicals arises from the Fenton reaction, where Fe (II) is oxidized by H2O2 to Fe(III), producing a
OH. Within cells, free radical production from Fe (II) is catalytic as Fe (III) is reduced back to Fe(II) at the expense of endogenous reducing species [211–213]. Fe (II) þ H2 O2 ! Fe (III) þ
OH þ OH ðFenton reactionÞ; Fe (III) þ O
 2 $ Fe (II) þ O2 ðredox reactionÞ; The net reaction (Haber–Weiss reaction); 
and O
 2 þ H2 O2 $ O2 þ OH þ OH

The iron-catalyzed Haber–Weiss reaction is considered to be a major mechanism by which the highly reactive
OH is generated in biologic systems [214, 215]. Nevertheless, in a lower grade, transition metal ions, such as copper, are also capable of catalyzing this reaction. Iron is clearly involved in the initiation of oxidative damage and formation of ROS, such as
OH or O
 2 . In addition, O
 combines with NO
to form ONOO , a 2 RNS, which is as detrimental as
OH in damaging proteins and DNA, and inhibiting DNA repair mechanisms [216].  O
 2 þ NO ! ONOO

Furthermore, NO binds reversibly and high affinity to Fe (II), either free iron, iron within iron–sulfur centers, or iron within hemoproteins, under physiological or 11

Romero et al. pathophysiological conditions [217]. During cellular respiration, the interaction of NO
and iron-containing enzymes has a high relevance [218]. NO
binds to iNOSheme iron and inhibits its activation. The dissociation of the Fe3+-NO complex competes with its reduction to the Fe2+-NO structure to inhibit iNOS activation [219]. Fe3þ þ NO
! Fe2þ þ NO ! Fe2þ  NO or Fe3þ þ NO
! Fe3þ  NO ! Fe2þ  NO Moreover, other reactions may also take place as a consequence of
OH generation, for example peroxidation of PUFA in lipids. The role of lipids in cellular membranes is not only structural but also functional; thus, any change in these molecules can lead to cell dead. If an excess of Fe (II) is present in the system, lipid hydroperoxides may undergo a Fe (II)-driven reaction [220–222]. Thus, iron is also a culprit in membrane phospholipid peroxidation; by the generation of oxygen radicals, Fe (II) induces lipid peroxidation. LH þ
OH ! L
þ H2 O L
þ O2 ! LOO
LOO
þ LH ! LOOH þ L
LOOH þ Fe (II) ! Fe (III) þ
OH þ LO
In summary, metal-mediated formation of free radicals may cause changes in DNA, protein and lipid structure or function and lead to changes in gene expression. As iron triggers oxidations via several reactions, protecting iron from molecular oxygen in the media is an essential event to avoid iron-mediated oxidations in biologic systems that may lead to abnormalities that affect cell or tissue functions which leads to serious cellular dysfunction or death. Obviously, a chelator molecule that binds iron would reduce reactions of free or labile iron with oxygen and its metabolites. Iron has six sites where a chelator can bind; thus, a hexadentate chelator such as desferrioxamine (DFO) completely deactivates free iron. DFO has shown high activity in Fe deprivation. However, some studies report DFO limitations; thus, its efficacy is severely limited due to its low ability to permeate biologic membranes [223–225]. Other hexadentate chelators are effective but far from being ideal therapeutic agents due to their low oral availability, toxicity, or iron donation to pathogenic organisms [226]. The ongoing therapeutic strategy to prevent metal-induced damage is the development of multifunctional iron-chelating compounds that modulate multiple disease targets [227–231]. Taking into account that antioxidants are the living organism’s major resource for protection against free radical-mediated damage, it would be reasonable to 12

develop an antioxidant therapeutic treatment line against iron damage (Fig. 5). Melatonin indirectly enhances antioxidant activity by promoting the activity of GPx [232, 233] and GRd [234] as well as mRNA levels of SOD [235]; also, GSH can act as a free radical scavenger [236]. Another reported antioxidative property of melatonin involves its diverse direct free radical scavenging activities [237–241]; the hydrophilic and lipophilic nature of melatonin may also be an important factor in this regard, allowing melatonin to move freely across all cellular barriers. These features and how iron generates free radical damage in an organism suggest that melatonin could play an important role against ironinduced damage (Fig. 5). Limson et al. [40] demonstrated, using adsorptive cathodic stripping voltammetry (ACSV), that melatonin forms in situ complexes with iron. Melatonin is able to interact with Fe (III) but not with Fe (II). Based on these data, they postulated that melatonin removes free Fe (III), thus preventing its reduction to Fe (II) avoiding free radical generation. Iron-mediated oxidative damage occurs in several neurological disorders [242–244]. Kabuto et al. [245] evaluated oxidatively destroyed tissue in rat brain homogenates after injecting FeCl3 to cause acute epileptiform discharges. After the injection, oxidative damage was evaluated by measuring levels of TBARS, which were obviously elevated. When melatonin (50–2000 lM) was co-incubated with FeCl3 (100 lM), lipid peroxidation was reduced in a concentration-dependent manner. Moreover, before rats were injected with FeCl3, pretreatment with melatonin (0.2 mmol/kg i.p.) limited TBARS concentration by 66% [245]. A quantitative study developed afterward showed that melatonin administration at pharmacological doses (5 mg/kg) for 5 days prior to and 1 day after intracortical injection of iron (ferric ammonium citrate 0.1 mM) reduced neuronal death by roughly 40% [246]. In patients with Parkinson disease, the iron content and lipid peroxidation of the brain are reportedly higher than in healthy controls [247]. Lin and Ho [248] evaluated melatonin’s neuroprotective actions on iron-induced neurodegeneration in the nigrostriatal dopaminergic system. Intranigral infusion of iron into rats caused degeneration of the nigrostriatal dopaminergic system, while coinfusion with melatonin (60 lg/lL) partially prevented iron-mediated elevation of lipid peroxidation. Additionally, in rat cortical homogenates, the in vitro activity of melatonin (0.1–4 mM) co-incubated with iron (ferrous citrate 1 lM) was also evaluated. As usual, melatonin in a dose-dependent manner overcame iron-induced lipid destruction. In a subsequent study, Chen et al. [249] used iron to induce oxidative damage in the locus coeruleus (LC) where degeneration has been documented in several neurodegenerative diseases. They observed that lipid breakdown was dose dependently elevated after iron infusion. Moreover, intraperitoneal administration of melatonin attenuated iron-induced lipid peroxidation in this nuclear group and prevented apoptosis and the reduction in locomotor activity. Studies in patients with AD revealed significantly augmented levels of iron in several brain regions [242,

Melatonin for protection against metals 250–252]. Ozcankaya and Delibas [253] reported lower melatonin levels and higher serum iron levels in patients with AD relative to these in patients with non-Alzheimer’s disease . Free radical damage was measured as MDA (the end-product of oxidative degradation of polyunsaturated fatty acids). Based on their results, both aging itself and AD promote lipid peroxidation. d-Aminolevulinic acid (ALA) is a heme synthesis precursor in some inherited and acquired porphyrias; it mainly accumulates in the liver. In the presence of Fe (II), ALA generates ROS, via metal-catalyzed oxidation to 4,5-dioxovaleric acid.
OH targets DNA, resulting in the formation of stable compounds of guanidine including 8-hydroxydeoxyguanosine (8-OH-dG), a specific and sensitive molecule, useful as an oxidative DNA damage biomarker. Qi et al. [254] found melatonin at concentrations >0.1 mM to reduce ALA-induced formation of 8OH-dG in a dose-dependent manner in calf thymus DNA. Melatonin was more effective than Trolox or mannitol in this system. They postulated that the highly effective protection of melatonin may be related to its direct
OH scavenging ability as well as to its ability to detoxify the precursor H2O2 [255]. Additionally, as rather high concentrations of melatonin occur in cellular nuclei [256, 257], the findings are consistent with the reduction in nuclear DNA damage. Other studies showed that melatonin at concentrations ranging from 1 to 100 lM inhibited 8-OH-dG formation and accumulation in a dose-dependent manner [205]. Thus, melatonin provides an effective protection against metal-induced DNA damage. One investigation using rat liver homogenates compared the antioxidative effects of melatonin with other nonindole antioxidants after incubation with FeSO4 and H2O2. Lipid peroxidation was measured as MDA and 4-HDA. Melatonin (5–16 lM) reduced MDA + 4-HDA levels induced by FeSO4, but it was somewhat less efficient than vitamin E or DFO but more effective than GSH or vitamin C. Melatonin exhibited synergistic effects in association with vitamins E and C and GSH as shown by the increased efficacy in reducing lipid peroxidation in rat liver homogenates [22]. A recent study, using an experimental biliary obstruction model in rats, reported the accumulation of iron in liver and blood. Also, the presence of loosely bound iron was related to oxidative stress induced by the biliary duct obstruction. Melatonin exerts a potent effect in regulating iron metabolism; this activity is also related to its action on loosely bound iron, lipid peroxidation, and tissue injury markers (alanine aminotransferase and alkaline phosphatase) in obstructive jaundice [258]. Preeclampsia is a hypertensive disorder that is associated with pregnancy, as a consequence of an imbalance between pro-oxidant and antioxidant defenses. A recent study proved that melatonin (250 lM) acts in human placental mitochondria as an inhibitor of NADPH- and irondependent lipid peroxidation (estimated from the quantity of TBARS formed), especially when combined with vitamin C (30 lM) and/or vitamin E (25 lM). In this in vitro study, melatonin markedly reduced TBARS formation in a concentration-dependent manner. When vitamin C and vitamin E were separately or collectively co-incubated with

melatonin, a greater effect was documented. In another study of Gitto et al. [22], there was also a synergistic action between melatonin and vitamin C, which may be related to the iron-chelating ability by melatonin. The authors postulate that, melatonin in combination with ascorbate and a-tocopherol may be a worthy treatment for preeclampsia; melatonin would permit a reduction in the doses of these vitamins and also strongly enhance their antioxidant effects in the placenta [259]. Adriamycin (doxorubicin) is a widely used anticancer drug with a high clinical efficacy. Due to its diverse toxicities, its use is limited. Adriamycin extracts iron from ferritin, creating an adriamycin–iron complex, which leads to lipid peroxidation [260]. Recently, Othman et al. [261] demonstrated that melatonin ameliorated oxidative stress caused by adriamycin by regulating iron levels. In this study where melatonin (15 mg/kg) was used before and co-currently with adriamycin, the inclusion of melatonin resulted in significantly decreased levels in iron levels in plasma compared with rats treated only with adriamycin. In each of these studies, melatonin exhibited a suppressive effect against iron-induced oxidative stress. Several processes may be involved in melatonin’s protection; (i) as a free radical scavenger [237–241], (ii) as an antioxidative enzyme activator [235, 248], and (iii) as an iron chelator [40]. Based on the published data, melatonin inhibits ironinduced endogenous oxidative injury in several tissues including the brain and liver, it may be useful as a treatment for pathologies for preeclampsia, and it also regulates iron levels increased by adriamycin. The protection afforded by melatonin supports the suggestion that melatonin may be an effective therapeutic tool in iron-induced oxidative stress. Copper and melatonin Copper, with other metals, has an essential role in several physiological functions after binding to specific proteins, participating in redox reactions, aiding the transport of oxygen and storage or transport of the metal itself. Copper is the third most abundant trace element in the human body; in biologic systems, it is found as copper (I) (Cu+) and copper (II) (Cu+2). Oxidation states of copper place this metal as a useful ion for redox reactions, functioning as an electron transfer intermediate. Copper’s catalytic activity as a cofactor in redox reactions is critical for several enzymes including SOD, ascorbate oxidase, ceruloplasmin, lysyl oxidase, cytochrome c oxidase, tyrosinase, and dopamine-b-hydroxylase and is required for functions such as cellular respiration, free radical defense, neurotransmitter synthesis, and neuronal myelination [262, 263]. Its capacity to participate in one electron change reactions makes copper both essential for life and toxic because it induces oxidative damage. Ceruloplasmin (CP) is the protein responsible for the transport of 95% of plasma copper. It is synthetized in liver where it binds to copper ions. Deficiencies in CP lead to an accumulation of copper in blood and also in several tissues. Unlike iron, copper has an effective excretion mechanism. Several proteins are involved in the 13

Romero et al. intracellular transport of copper; metallochaperones are metal receptor proteins responsible for the transport of copper to its intracellular destination and they also protect this ion from reactions at a multitude of alternative binding sites [264]. Inside the cell it is distributed by specific chaperones. When in excess, metallothioneins are induced which then sequester the excess copper [265, 266] (Fig. 5). Copper can catalyze ROS formation via Fenton and Haber–Weiss chemistry [267] and can induce oxidative stress by two mechanisms: (i) copper can directly catalyze the formation of ROS via a Fenton-like means [267, 268]. The cupric ion, Cu(II), in the presence of O
 or 2 biologic reductants such as ascorbic acid or GSH, can be reduced to cuprous ion, Cu(I), which catalyzes the formation of the highly reactive
OH through the decomposition of H2O2 via Fenton reaction [269]. (ii) Exposure to elevated levels of copper depletes GSH concentrations [270]. Disruption of copper homeostasis resulting in elevated pools of copper may contribute to a shift in redox balance toward a more oxidizing environment due to the depletion of GSH levels [271]. A reduction in GSH may enhance the cytotoxic effect of ROS and allow the metal to be more catalytically active, thus producing higher levels of ROS. On the other hand, antioxidant defenses can be directly or indirectly compromised under conditions of a copper deficiency; a loss of CP, selenium-dependent GPx, Cu/Zn-SOD, and catalase activity as well as a rise in GSH and metallothionein has been observed [272]. Cu (I) þ H2 O2 ! Cu (II) þ
OH þ OH ðFenton reactionÞ Cu (II) þ O
 2 $ Cu (I) þ O2 ðredox reactionÞ Also, copper induces, via oxygen free radicals, the oxidation of bases and causes DNA damage; in both states, copper is more active than iron and induces DNA breakage [273]. Under in vitro conditions, copper exerts higher oxidative damage than iron mainly due to copper’s ability to bind proteins nonspecifically [274]. Moreover, Rae et al. [275] confirmed that free copper within the cells is limited to less than one free copper ion per cell, suggesting that cells are capable of maintaining copper in a bound state, thereby avoiding the formation of free radicals. Indeed, when iron and copper chelators were used to prevent H2O2-induced DNA damage, only iron chelators were effective, indicating that the main redox-active transition metal inside the cell is iron [276]. Copper overload due either to a high dietary intake or to transport alterations can result in many well-known disturbances, such as Wilson’s or Menkes disease. An imbalance between copper absorption and excretion can increase copper levels, and cells may not be able to maintain all the copper in the protein-bound state, mediating free radical production and oxidation of lipids, proteins, and DNA, causing impaired cellular functions and eventually cell death [263, 277]. Copper toxicity is related to its pro-oxidant activity. It is well accepted that copper can irreversibly and nonspecifically bind to thiol and amino 14

groups in proteins [278]. In addition, alterations of copper distribution are associated with neurodegenerative diseases, including AD, amyotrophic lateral sclerosis, PD, prion diseases, and Huntington’s disease [279–283]. Markers of elevated oxidative stress have been documented in a variety of tumors, possibly due to the combination of factors such as elevated active metabolism, mitochondrial mutation, cytokines, and inflammation [284]. Elevated copper levels have been shown to be directly linked to cancer progression [285]. Copper is important also for angiogenesis. Moreover, copper is known to bind to Ab via histidine and tyrosine residues [286]. Cu(II) interaction with Ab promotes its neurotoxicity which correlates with the metal reduction [Cu(I)] and with the generation of oxidative stress contributing to AD progress. The copper complex of Ab (1–42) has a highly positive reduction potential, characteristic of strongly reducing cupro-proteins. Abnormal levels of copper are also linked with diabetes [287], atherosclerosis [288], and cardiovascular disease [289]. Ab-Cu(II) þ AscH $ Ab-Cu(I) þ Asc þ Hþ Ab-Cu(II) þ Asc $ Ab-Cu(I) þ Asc Ab-Cu(I)þH2 O2 $Ab-Cu(II)þ
OHþOH (Fentonreaction)

Ab-Cu(I) þ O2 $ Ab-Cu(II) þ O
 2 As with other metals, some in vitro studies using electrochemical methods indicate that melatonin forms complex with copper, exhibiting a concentration-dependent affinity [40, 290]. This means that melatonin, besides acting as a potent free radical scavenger, may also bind copper preventing it from participating in free radical generation in vivo. In these studies, it was not possible to determine whether this was a simple ligation or a chelation. There is an interesting relationship in fish exposed to the copper concentrations of the water and a loss of the circadian locomotor rhythm; copper did not impede the secretion of melatonin, but the loss of locomotor activity implied that fish were not able to respond to it probably due to melatonin interacting with copper [291]. Also, Parmar and Daya [292] studied the effect of copper on pineal indoleamine synthesis and found that melatonin, when coadministered with copper, prevented N-acetyltransferase inhibition induced by copper. Parmar et al. [290] examined copper-induced lipid peroxidation in rat liver homogenates and concluded that coincubation of copper and melatonin affords protection against associated free radical damage. Wakatsuki et al. [293] determined the effects of melatonin in copper-mediated low-density lipoprotein (LDL) oxidation in normolipidemic postmenopausal women, concluding that melatonin may protect LDL against oxidation. There are other reports indicating that melatonin inhibits in vitro Cu

Melatonin for protection against metals (II)-induced LDL oxidation [294–296]. As free radicals may induce oxidative modification of LDL, a process believed to be involved in atherogenesis, the authors summarized that melatonin can inhibit oxidative modification of LDL because of its free radical scavenging effect and its interaction with copper as well. Mayo et al. [297] investigated the protective effect of melatonin, compared with other antioxidants, against oxidative protein damage induced by metal-catalyzed reactions. They concluded that melatonin was more effective and consistently protected against the structural damage caused by Cu (II)/H2O2 than other antioxidants (Trolox, ascorbate). Numerous reports have documented the protective actions of melatonin on DNA and lipids; in this article, the authors also demonstrated the effectiveness of melatonin in protecting proteins from peroxyl radicals. Zatta et al. [298] investigated the action of melatonin in free radical formation due to the interaction between b-amyloid peptides and metal ions. In this work, the authors showed that various b-amyloid peptides produce a relatively modest quantity of free radicals in the absence of metal ions. This production is greatly increased in the presence of Cu(II) and Fe(II). Furthermore, melatonin had a large inhibitory effect on free radical production. The authors concluded that melatonin may have great pharmacological potential for reducing the free radical production produced by the interaction between b-amyloid peptides and metal ions. In conclusion, the characteristic electronic configuration of copper provides its chemical properties favoring its biologic functions but also confers to copper the ability to generate excessive amounts of ROS. Copper overload toxicity due to a redox imbalance can be addressed by scavenging free radicals or chelating the excessive copper, and melatonin may fulfill these requirements, thereby providing protection against copper toxicity. Removal of excessive copper from proteins such as CuZn-SOD can have negative consequences; therefore, copper chelation therapy should be directed specifically to the site or tissue of interest. Nickel and melatonin Although a trace element for plants, some bacteria, and invertebrates (it is the central metal of ureases), nickel is toxic to humans. Nickel is not an abundant element on Earth, but both industrial and social uses, such as electroplating, and its use as anticorrosive, in batteries, jewelry, or decoration, have stimulated its use. Nickel passes into the atmosphere, water, and soil from the burning of fossil fuels, metal foundries, or tobacco smoke. Humans take in nickel by inhalation, dermal absorption, or ingestion of contaminated food. It is important to note that vegetables grown near to dense traffic roads present high concentration of nickel [299]. Nickel toxicity greatly depends on what chemical species are formed. Nickel salts are not that toxic, but soluble Ni2+ cations are highly toxic, although they are eliminated from the body easily [300]. Nickel particles target mainly the respiratory tract, leading to respiratory pathologies and cancer. Indeed, nickel species induce tumors in several animal models and sites of application [301]. The most

carcinogenic species of nickel are the disulfides and oxide dusts, nickel sulfate, nitrate, and chloride, and the vapor containing carbonyl nickel [302]. Tetracarbonyl nickel [Ni (CO)4] because of its high toxicity, penetration into cells, and persistence deserves special consideration. Regarding the biochemical behavior of nickel, it easily penetrates into cells where it binds low molecular weight proteins. Ni2+ selectively blocks T-type voltage-dependent calcium channels (VDCC) (at 50 lM), but at higher concentrations, it blocks all types of VDCC. Interestingly, at lower concentrations than those blocking VDCC, Ni2+ enters to the cell through VDCC, affecting intracellular Ca2+ homeostasis [303]. From a toxicological point of view, Ni2+ reportedly damages DNA by ROS generation derived from the catalyzed Fenton reaction, which preferentially forms H2O2, and by activating pro-inflammatory signals such as the transcription factor NF-jB. This is also related to skin hypersensitivity elicited by nickel complexes [304]. DNA damage suffers depletion of its supercoiled structure. Furthermore, DNA repair systems are disrupted by nickel, contributing to the total toxic effect through a mechanism that implicates the binding to DNA repair enzymes and the subsequent protein degradation [305]. Allergic contact dermatitis, triggered by skin exposure to Ni2+, is well accepted as being the result of the formation of organometallic complexes between nickel and electron-rich ligands [306], basically nucleophilic amino acids, generating the subsequent antigen. The oxidative stress scenario elicited by nickel is not only responsible for its carcinogenic effect, but also it is known that lipid peroxidation occurs in human plasma when nickel chloride is administered [307]. Another important target of nickel is HIF-1, as noted for other metals [308]. Nickel would displace iron from the oxygen carrier of hemoglobin, that is, hemo group, what induces a permanent hypoxia, thus activating HIF-1 [309]. Studies have considered the role of nickel in neurotoxic processes affecting cultured neurons and cell lines in relation to the protective effect of melatonin. Nickel-triggered oxidative stress and ROS generation in cortical neurons and neuro-2a neuroblastoma cells are mitigated by incubation with melatonin at millimolar concentrations [310]. Incubation of nickel chloride at concentrations from 0.125 to 1 mM produced a dose-dependent increase in ROS, measured with the fluorescent dye DCFH-DA, in both cortical neurons and neuro-2a cells; this was more apparent in the primary cultures, as ROS generation was enhanced up to threefold in the presence of 1 mM Ni2+, while neuro-2a cells only exhibited a 1.7-fold augmentation of ROS at the same concentration of Ni2+. Under similar experimental conditions, mitochondrial O
 con2 centrations, measured with the redox-sensitive dye MitoSOX Red, were elevated along with increasing concentrations of Ni2+ [311]. In this scenario, cell viability, measured with the method of the MTT reduction, was reduced during exposure to increasing concentrations of nickel, with a maximal reduction at 1 mM Ni2+ (by 50% in both cell cultures). Under these conditions, incubation with 1 mM melatonin dramatically reduced ROS generation, as well as mitochondrial O
 concentrations. 2 15

Romero et al. Melatonin prevented Ni2+-induced ROS production when [Ni2+] was between 0.125 and 0.5 mM. Ni2+-elicited ROS production was partially diminished by 1 mM melatonin in cortical neurons; ROS levels were increased by 50% over those in cells not exposed to melatonin. Better results were found in neuro-2a neuroblastoma cells where melatonin exerted a complete prevention of ROS generation in the presence of 1 mM nickel. Indeed, no significant differences were found between control (Ni2+) and sample (nickel plus melatonin) cultures. The mitochondrial O
 2 concentration was reduced by approximately 50% when 0.125, 0.25, or 0.5 mM of Ni2+ was applied in the presence of 1 mM melatonin for 24 hr [311]. Pretreatment with melatonin resulted in similar results after different times of exposure to Ni2+, from 3 to 24 hr in both cell cultures [310]. As far as the loss of cell viability due to exposure to Ni2+, 1 mM melatonin, counteracted the neurotoxic actions with maximal protection in the presence of 0.5 mM of Ni2+. At higher concentrations of Ni2+ (1 mM), melatonin only rescued 50% of the cells from death for cortical neurons and neuro-2a cell cultures. Melatonin exerted the neuroprotection over a wide Ni2+ exposure times, from 3 to 24 hr. The protective actions of melatonin in these studies were presumably related to the enhancement of mitochondrial functions, for example maintaining membrane potential, increased ATP synthesis, and stabilization of mitochondrial DNA. To confirm these suppositions, confocal microscopy and flow cytometer were performed. JC-1 dye was used to assess the preservation of the mitochondrial membrane potential. Loss of red and increase in green fluorescence accounted in JC-1-charged cortical neurons treated with Ni2+ at 0.5 and 1 mM for 12 hr, and pretreatment with melatonin preserved that loss of membrane potential. In addition, ATP content was analyzed by an ATP determination kit; DNA was reduced in both cell cultures when Ni2+ was administered at concentrations from 0.125 to 1 mM; the pretreatment with melatonin at 1 mM preserved the ATP content in the presence of all nickel doses investigated [310, 311]. The assessment of the mitochondrial DNA integrity was evaluated by quantitative real-time PCR. A marked reduction in mtDNA became apparent when cortical or neuro-2a cells were exposed to nickel, at concentrations between 0.25 and 1 mM. Pretreatment with 1 mM melatonin prevented mtDNA reduction and elevating mtDNA concentration up to the level of the control situation. Nickel also provoked a marked reduction in the mtDNA transcription, which was efficiently counteracted by melatonin in the presence of all Ni2+ concentrations evaluated. In addition, 8-OHdG was used as a biomarker of mtDNA oxidative damage in neuronal cells with an enhancement of 8-OHdG-positive cells in the presence of increasing concentrations of Ni2+. Similarly, these increases were attenuated by 1 mM melatonin, being particularly efficient when Ni2+ concentrations were 0.125 and 0.25 mM [311]. Finally, the mtDNA nucleoid structure impairment elicited by Ni2+, which causes disruption of DNA functionality in terms of replication/transcription regulation, DNA content, and DNA repair systems, was abolished by pretreatment with 1 mM melatonin. However, melatonin only reduced nucleoid structures in the presence of 0.5 mM Ni2+. These 16

results confirm the higher vulnerability of mtDNA to nickel with respect to nuclear DNA. Also, mitochondria are key targets of nickel toxicity. Hence, melatonin, due to its potent antioxidant properties which included its ability to scavenge Ni2+-triggered ROS, is an important antidote against Ni2+ exposure [310, 311]. Cobalt and melatonin Cobalt is a trace element in biota, where it is presented as trivalent and divalent ions, forming a huge number of organic and inorganic salts. In nature, cobalt is found together with other metals such as copper, manganese, nickel, and arsenic. Cobalt is mobilized into the atmosphere and water doing the burning of coal and oil, exhaust from engines, and industrial waste. Industrial uses of cobalt include as a paint additive and radioligand in nuclear medicine [28]. Humans can be intoxicated with cobalt during job exposure or overmedication of cobaltcontaining drugs, such as cobalamin preparations. Clinical symptoms of cobalt intoxication include allergies [312] and blood disorders [313], among others. Cobalt fulfills an essential role in humans as an integral part of vitamin B12. Moreover, cobalt is related to the formation of thyroid hormones [314]. However, when an excess of free cobalt occurs, cytotoxic signals are triggered, mainly derived from a free radical overgeneration [28]. Also, Co(II) carcinogenicity has been demonstrated in animal studies [315]. Cobalt inhibits DNA synthesis [316] and causes DNA damage and DNA-protein cross-linking, as well as disrupting the DNA repair system [317]. It has been reported to inhibit mitosis [318] and reduce platelet aggregation [319]. High concentrations of cobalt promote various protein modulations, for example inhibition of delta-aminolevulinic acid synthase [320], activation of arginase, and induction of acylamino acid hydrolase [321]. Also, cobalt depletes neurotransmitters from several regions of the brains [322]. Mitochondrial DNA is also target for cobalt salts [323]. Global alterations in cell physiology induced by Co(II) is similar to that described for hypoxia, as the HIF-1 alpha was found to be up-regulated, as a means of preventing its ubiquitination and proteasomal degradation [324]. In terms of neurodegenerative diseases, one of the most important hypotheses to explain AD on set and progression is the disequilibrium in brain metal concentrations, mainly focused on copper and zinc [325]. Also, however, cobalt concentrations were found to be elevated in postmortem brain tissue of patients with AD [252, 326]. Co(II)-induced free radical generation seems to depend on metal chelation, as absence of GSH or histidine reduced Co(II) efficiency [327]. This finding could be the clue to explain the protective role of melatonin against cobalt-induced free radical generation, taking into account its confirmed metal-chelating properties [40], without discarding the radical scavenging feature described for melatonin. Oliveri et al. [319] noted that SH-SY5Y neuroblastoma cells exposed to increasing concentrations of cobalt chloride induced a concentration-dependent drop in GSH levels. This reduction was prevented by 1 lM

Melatonin for protection against metals melatonin. Both the antioxidant and chelating activities of melatonin were the proposed mechanism for the protective activity of the indoleamine on GSH levels. Melatonin activates the rate-limiting enzyme in GSH synthesis, that is, gamma-glutamylcysteine synthase [328]. Melatonin also preserved the viability of cells exposed to cobalt. Furthermore, 0.3 mM CoCl2 produced a 1.5-fold increase in the Aß release from SH-SY5Y cells. This rise was not observed in Co(II)-treated SH-SY5Y cells pre-incubated with melatonin [319]. The amyloidogenic feature of cobalt may be due to its inhibition of PKC, taking into account such inhibition favors amyloid precursor protein (APP) processing toward ß-secretase-addressed Aß formation. Inhibition of PKC was presumably due to competition with Ca2+, thus reducing the activity of this Ca2+-dependent enzyme. Although the exposure to metallic cobalt particles would seem to have the most direct relationship to tumor cell proliferation, soluble cobalt salts also relate to physiopathological signals involved in angiogenesis such as vascular endothelial growth factor (VEGF), which is upregulated by HIF-1, a key mediator of the hypoxia response. As mentioned above, HIF-1 exhibits enhanced activity due to exposure to cobalt [329]. Melatonin not only diminished VEGF protein and mRNA synthesis under basal conditions, but also this reduction is more pronounced in the presence of 0.1 mM CoCl2, which drastically increased both VEGF protein and mRNA production. 1 mM melatonin abolished completely the elevations in various tumor cell lines [329]. Similar observations were found in HIF-1 expression, as melatonin counteracted the CoCl2-promoted HIF-1 overexpression. Interestingly, melatonin does not affect basal levels of HIF-1. The proposed mechanism of the VEGF underexpression has its origin in the Co(II)-induced HIF-1 inhibition. Vanadium and melatonin Vanadium is trace element essential for many living organisms, including humans. It plays several roles in biota, including as counterions of proteins, DNA, and RNA. Sufficient doses of vanadium for humans are very small, so environmental or labor exposure to vanadium species easily leads to an exceeded concentration causing toxicity [330]. Vanadium is mobilized and gets to natural beds and the atmosphere from the combustion products of oils, dusts, and fumes. Accumulation of vanadium is mainly found in liver, kidney, and bones [28]. Vanadium compounds are used in chemotherapy [331], due to their inhibitory action of xenobiotic enzymes and cancer cell metastatic potential. Other uses of vanadium compounds are as a diabetes therapy, as a contraceptive and as nutritional complement [332]. The principal vanadium species is pentavalent vanadate, which can be reduced by hydrogenases to vanadyl (IV) species; when this happens, NADPH or GSH levels are lost and H2O2 is formed. Vanadate species are capable of generating the
OH and other ROS. Vanadium-induced ROS generation triggers several signaling pathways, influenced by proteins such as AP-1, MEK-1, and ERK-1. [333]. Further oxidative stress scenarios can be achieved

from its demonstrated inhibition of tyrosine phosphatases [28], and this causes loss of tyrosine phosphatase substrates, for example Na+/K+ ATPase and Ca2+-dependent ATPases. Final consequences of these activations are huge, but DNA damage is the most obvious. Like cobalt, vanadate species up-regulate the HIF-1 protein, inducing its expression through the PI3K/Akt pathway [334]. Vanadyl (IV) compounds also present free radical-derived toxicity, and this oxidative stress signal leads to programmed cell death [335]. In an isolated pulmonary arterial ring preparation, designed to measure the contractile effect of vanadate, presumably due to H2O2 overexpression, this anion evoked an inotropic response at 0.1–1 mM concentration that was efficiently annulled by 1 mM melatonin. Vanadium-elicited NADH oxidation was reduced by melatonin in a concentration-dependent fashion, as increasing concentrations of melatonin reduced the oxidation of NADH up to a 75% [336]. As previously noted, therapeutic use of organic chelated vanadium compounds includes as a diabetes treatment. This is because vanadium has been found to possess insulin-mimetic properties, as well as enhancing the effects of insulin when administered orally [337]. However, overuse of vanadium-derived drugs to treat diabetes leads to toxicological phenomena, preferentially at the kidney level. Administration of organic chelated vanadium resulted in a decrease in the mitochondrial membrane potential, while pre-incubation with melatonin attenuated this decrease. Furthermore, vanadium-generated hydroxylated free radical species in renal tubules were reduced in preparations including melatonin. In general, co-administration of melatonin with the vanadium-derived drugs produced a reduction in serum glucose levels indicating that melatonin can have a beneficial effect in patients with diabetes treated with vanadium compounds, mainly because of its protective role on renal function [337]. Molybdenum and melatonin Molybdenum presents different oxidation states with an easy redox transfer. It occurs on Earth preferentially as a sulfured mineral (molybdenite, MoS2). Industrial uses of molybdenum include its uses in the manufacture of hightemperature-resistant alloys, catalysts, lubricants, and dyes [299]. Molybdenum is a trace element for humans and it is necessary in very low doses. It is a key component of several enzymes referred to as molybdenum hydroxylases or molybdoenzymes, which participate in the biotransformation of xenobiotics [338], that is, aldehyde oxidase, sulfite oxidase, xanthine dehydrogenase, and xanthine oxidase. It also plays a role in the metabolism of purine. The molybdoenzymes contain FAD, two iron–sulfur centers, and hexavalent molybdenum in the form of a pterin cofactor. Thus, molybdenum oxidizes substrates and is reoxidized by molecular oxygen through a mechanism involving iron centers [299]. Overexposure to molybdenum can lead to toxicity scenarios. High concentrations of molybdenum induce the molybdoenzyme xanthine oxidase overexpression. Taking 17

Romero et al. into account they use molecular oxygen to reoxidize the molybdenum-centered cofactor, their overexpression leads to a higher generation of ROS, for example O
 and 2 H2O2. Hence, molybdenum toxicity is well correlated with the overactivity of xanthine oxidase and related enzymes, that is, during ischemia–reperfusion injuries in heart, skeletal muscle, and brain [339]. Sulfite oxidase, which reportedly does not produce ROS, has been linked to the some neurological defects [339]. Chronic exposure to molybdenum provokes high uric acid levels, loss of appetite, diarrhea, anemia, and a slow growth rate. High molybdenum density areas have been related to the appearance of gouty diseases [340]. In summary, molybdenum toxicity is indirectly related to ROS generation, as molybdoenzymes overexpression seems to be responsible of the oxidative stress-derived injury when excessive molybdenum is present. Only a few contributions discuss the role of melatonin in the modulation such as molybdenum-triggered damage [337]. The reason for this lack of work in this area may be the tricky chelation of the high valence molybdenum species present in biota, for example molybdate (MoO2 4 ), by potential ligands such as melatonin. It is accepted that ingestion of molybdenum, and before incorporation as a center metal of molybdenum hydroxylases, molybdenum is retained in the liver linked to a nonprotein cofactor bound to the mitochondrial outer membrane. In this state, melatonin, by chelating molybdenum, could compete with the transferring apoenzyme. However, the potential organometallic complex with melatonin does not seem to be possible because of spatial and electronic issues. Thus, melatonin may not be capable of exerting protective actions against molybdenum-induced toxicity. Exploring the chelating properties of melatonin: A proposed hypothesis Melatonin is a nonenzymatic antioxidant (namely antioxidant H) that is able to counteract the oxidative actions of metals by two major means, that is, scavenging the generated ROS and capturing such metals to form chelates, thus deactivating their ability to trigger oxidative stress processes [341]. Physicochemical and theoretical studies have focused on the antioxidant properties of melatonin, extensively reviewed by Galano et al. [342]. Radical scavenging drives the formation of several metabolites including cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N-acetyl-5-methoxykynuramine. By contrast, the corroborated chelating properties of melatonin [40] are not that simple to explain from a chemical point of view, as indole nitrogen does not have the ability to complex electrophile elements, taking into account its lone pair contributes to the aromaticity of the heterocyclic nucleus of melatonin. It is worthwhile mentioning that any metal, proposed to be chelated by melatonin, would present its own requirements to achieve such a chelating complex, following organometallic rules for the ligand–metal coordination, namely the 18 electrons rule (16 electrons for those called ‘platinum metals’, e.g., Ni2+ complexes) and the generation of tetrahedral, octahedral, or square planar-like 18

structures. For all of these reasons, a high possibility exists that the p-electrons of the benzene-fused ring are recruited to smoothly bind the metal, in a g6-hapticity fashion, depending on the number of electrons necessary to form a stable organometallic complex. Thus, a hypothetical representation could be as schematized in Fig. 2.

Concluding remarks This review summarizes the impact of metal-induced free radical formation on cells and how melatonin may counteract its toxic effects. Metals induce ROS/RNS generation which triggers epigenetic changes, abnormal cell signaling, uncontrolled cell growth, initiation of cellular injury, and the stimulation of inflammatory processes. Melatonin constitutes a valuable protector versus metal-induced damage due to its multiple properties: (i) direct free radical scavenger, (ii) increasing activity and expression of antioxidant enzymes, (iii) high lipophilicity which makes its easy transport across cellular membranes, (iv) chelating activity, and (v) its low toxicity. Certainly, after the analysis of the studies carried out in animal models and in vitro systems performed, it can be concluded that melatonin is a promising therapeutic tool for metal-related damage. However, further studies are necessary to confirm the usefulness of melatonin against metal-induced toxicity. Nevertheless, the realization of clinical trials on the possible usefulness of melatonin reducing metal-induced toxicity may result in promising data for several diseases that involve metals in their physiopathology.

Acknowledgements C. de los Rios thanks IS Carlos III for research contract under Miguel Servet Program. We thank Eva M. Garc
ıaFrutos (Instituto de Ciencia de Materiales, CSIC, Madrid, Spain) for co-crystallization of melatonin with selected metal.

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