Metallothionein 2A gene polymorphisms in relation to diseases and trace element levels in humans

Metallothioneins are a superfamily of cysteine-rich, intracellular, metal-binding proteins present in plants, vertebrates, invertebrates, eukaryotes, and prokaryotes. Historically, the discovery of MTs has closely been related to the study of Cd. The earliest work in this area was reported in 1941 by Maliuga, whereas the data on Cd-binding protein isolated from equine renal cortex, later named metallothionein, were first reported by Margoshes and Vallee in 1957 (reviewed in 5–9). Since then, MT has been of great interest in many scientific disciplines, including toxicology, biological and physical chemistry, molecular biology, and various clinical and cancer studies with about 10,000 published papers (7, 8, 9). The characteristics of MTs are low molecular mass of 6–7 kDa, high cysteine content (about 30 %), no aromatic amino acid, and high binding affinity for metals, particularly for Zn, Cu, and Cd. The amounts and ratios of metals bound by the thiol (–SH, mercaptide) group will depend on the tissue; human liver MTs mostly contain Zn and small amounts of Cu, while renal cortex MTs mostly contain Cd, then Zn, and then Cu (reviewed in 5, 8–12).

In mammals, including humans, there are four main groups of MTs with different sequences, expression, and characteristics: MT1, MT2, MT3, and MT4. Isoforms MT1 and MT2 are expressed in almost all tissues. MT3 is expressed mainly in the brain, and to a lesser extent in the heart, kidneys, and reproductive organs (reviewed in 8–10, 13–15). MT4 is expressed in the epithelial cells of the skin and mucosa (16). MT molecules are single-chain polypeptides, which contain 61 to 68 amino acids, and 20 of them are cysteine, ordered in the sequences Cys-Cys, Cys-x-Cys, and Cys-x-y-Cys (x and y are amino acid which are not cysteine) (8, 9, 10, 17). Cysteine sulphur atoms are responsible for binding divalent metals in two clusters of MTs, connected with a sequence which does not contain cysteine. Amino acids 1 to 30 form a stable α-cluster (C-terminal) with four metal binding sites, whereas amino acids 31 to 68 form a reactive β-cluster (N-terminal) with three binding sites for divalent metals ions. Therefore, each MT molecule can bind up to seven divalent ions of Zn, Cd, Hg, and Pb, 12 of Cu, and 18 of Ag. Four metal ions first fill the α-cluster, and remaining three ions enter the β-cluster. Metals bound to the β-cluster are released more easily than metals bound to the α-cluster (8, 10, 13, 15, 18). The order of metal-binding affinities was tested by in vitro studies on rat liver in the 1980s and the reports are not uniform: Cd >Pb >Cu >Hg >Zn > A g >Ni = Co ( 1 9 ) , Hg>Cu>Cd>Zn>Ni=Co (20), and Hg>Ag>>Cu>Cd> Zn (21). Many metals have the affinity for MT but only Cu⁺, Cd²⁺, Pb²⁺, Ag⁺, Hg²⁺, and Bi²⁺ can displace Zn²⁺ in MT, which was confirmed in the horse kidney in vitro (22) and in the rat liver in vivo studies (23). Exchanges between Zn and Cd happen rapidly in the β-cluster, contrary to their slow exchange in the α-cluster. Zinc readily dissociates from MT to make itself available for different biological functions and to stimulate further MT synthesis. Metallothioneins serve as metal ion donors to other ligands or proteins (reviewed in 13, 24, 25). Their degradation depends on metals bound, their distribution in MT molecules, and the medium. Acidic media are known to speed up metal dissociation from MTs. MTs completely saturated with metals are more resistant to degradation by lysosomal proteases than unsaturated MTs or apothioneins (apo-MTs, that is MTs free of metals). In neutral media, MTs saturated with metals are less resistant to degradation than apo-MTs (25, 26, 27, 28).

The synthesis of MTs is induced by numerous factors such as metals and metalloids, various chemical agents, including acetaminophen (paracetamol), cytokines, and many other stress-producing conditions, including oxidative stress, infection and inflammation. The peculiar chemical structure of MTs gives them their molecular stability and specificity and defines their role in various physiological and pathological conditions (8, 9, 10, 11, 29, 30, 31, 32, 33). Their main biological function is to maintain the homeostasis of essential metals Zn and Cu (reviewed in 12). Studies conducted in vitro showed reactivation of apo-enzymes in which Zn or Cu were cofactors (alkaline phosphatase, superoxide dismutase and others) after incubation with Zn-MT or Cu-MT. The mechanism of Zn donation from MT to apo-enzyme is still unknown, but it is assumed that MT binds to an MT-releasing factor, which displaces Zn and makes it available to enzymes (8, 14). It has been shown that MTs participate in Zn regulation by intestinal absorption and excretion. When Zn intake is high, MTs may have a crucial role in restricting its absorption by storing it in the enterocytes and enabling its transfer back to the gut lumen, as confirmed by studies on knockout and transgenic mice (34, 35, 36, 37, 38, 39). The induction of intestinal MT by Zn and its interaction with Cu is used in the therapy of patients with Wilson’s disease with Zn acetate, a US Food and Drug Administration-approved drug. Wilson’s disease is a rare autosomal recessive inherited disorder of Cu metabolism characterised by the accumulation of excessive amounts of Cu in the liver, brain, and eyes. The mechanism of Zn action as an anti-copper agent involves inhibition of both Cu absorption from the gastrointestinal tract and its transfer into the circulation by capturing the Cu-MT complex in the mucosal cell and its ultimate faecal excretion (40, 41).

Metallothioneins have multiple roles. Besides its main function to keep essential elements in balance, they protect the body against free radicals and toxic effects of metal ions (reviewed in 8–10, 13, 24, 42−46). High levels of MTs can be found in foetal and neonatal liver, but these drop to the levels found in adults during the postnatal period. Increased liver MT levels during prenatal period in all mammalian species are believed to protect against potentially toxic Zn and Cu ions before the intestinal control mechanisms develop (46, 47, 48, 49). Another important role of MTs is to protect against oxidative stress caused by various environmental stressors, including toxic metals. Experimental studies showed lower acute hepatotoxicity of Cd due to induced MT synthesis and high Cd binding to cytosolic MT, which reduces exposure of target organelles to Cd (reviewed in 32). Studies conducted on knock out mice showed that those without MT expression were more sensitive to Cd toxicity than control mice. The protective effects of MTs are generally clear against acute metal toxicity and carcinogenicity but not as much against chronic metal toxicity, to be addressed later in the text (8, 50, 51, 52). In general, large amounts of –SH groups in MT molecule enable reaction with numerous electrophilic chemicals, as they catch free radicals such as hydroxyl, superoxide or nitric oxide radicals produced during metabolism of xenobiotics (33, 53, 54, 55, 56, 57).

Other important roles of MTs involve cell survival, inhibition of apoptosis, angiogenesis and vascular remodelling, and immunomodulation. Studies on human umbilical vascular endothelial cells (HUVECs) have shown that a homedomain protein HMBOX1, which acts as a transcription factor and is abundantly expressed in the cytoplasm of the endothelial cells, maintains cell survival by promoting autophagy and inhibiting apoptosis by interaction with MT2, which increases intracellular free Zn (58). This role of MTs in vascular remodelling is important in the development of atherosclerosis and malignant tumours. Furthermore, MTs seem to inhibit pro-inflammatory cytokines, such as interleukins IL-6 and IL- 12 and tumour necrosis factor TNF-α, and can therefore supress inflammation (59). Investigations on MT-null mice showed higher susceptibility to the hepatotoxic effects of the anti-inflammatory drug paracetamol (acetaminophen), which points to the protective role of MTs against chemically induced hepatotoxicity (60, 61). The protective antioxidant role of MTs against radiation-mediated immunosuppression and cell damage was confirmed in experiments on MT-null mice (62, 63).

Synthesis of MTs in humans is encoded by a cluster of genes located in the q13 locus of chromosome 16 (16q13). Until now, 17 genes have been identified in this cluster, and at least 11 of them are functional; eight among MT1 isoforms (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT 1M/MT1K, and MT1X), and the other three have only one functional gene (MT2A, MT3, and MT4) (reviewed in 8–12, 15, 45, 64–67). The genes consist of two to three exons and one to two introns. Elements that control MT transcription can be divided in basal and inducible. The basal elements of gene sequence are the TATA-box, GC-box, and at least two basal level enhancer (BLE) sequences. The promoter region of the MT1 and MT2 genes involve inducible elements that consist of different types of responsive elements: metal response elements (MREs), glucocorticoid response elements (GREs), and antioxidant response elements (AREs). The most investigated mechanism of MT gene transcription by metal ions is via several MREs located in 5’ untranslated region (UTR) of the gene (67, 68, 69). Early studies showed that metal transcription factor 1 (MTF-1) binds to MREs in the promoter regions of MT genes via Zn finger transcription (Cys2-His2) factor controlling the expression of the MT1 and MT2 genes. Besides Zn, MTF-1 can be activated by reactive oxygen species, tyrosine-specific protein kinase, protein kinase C, and c-Jun N-terminal kinase (68, 69, 70, 71, 72).

Metal ions other than Zn can induce MT synthesis by mechanisms different than the one described above. Toxic metals cannot activate MTF-1 and, due to high binding affinity for MT, they replace Zn ions in MT molecules and thus increase intracellular Zn levels (reviewed in 13–15, 24). Free Zn then stimulates further synthesis of MT by binding to MTF- 1, which then binds to MRE and ultimately has impact on metal toxicity. In other words, under conditions of acute exposure to high doses of toxic metals such as Cd or Hg, higher MT expression may reduce their toxicity. However, in chronic exposure to either of the toxic metals (Cd or Hg), increased MT synthesis leads to prolonged retention of that metal in the body, which increases the risk of toxic effects. In addition, increased MT may capture essential elements in internal organs, primarily Zn in the liver, making them less available for their physiological roles such as transfer to the developing foetus through placenta during pregnancy (reviewed in 8, 73).

Metallothionein expression can be induced by oxidative stress when generated hydrogen peroxide (H₂O₂) radicals oxidise MT, and Zn is released, which then activates MTF-1 (64). Glucocorticoids also regulate MT transcription by binding to their response elements (GREs) in the promoter region of the MT genes (74). MT expression can be also induced also by tissue hypoxia (75), catecholamines (76), or hypothermia (77).

Single nucleotide polymorphisms (SNPs) are genetic variations characterised by the replacement of one nucleotide with another in a certain stretch of DNA, which occurs in at least 1 % of the population and differs between population groups. Given their location, SNPs can either be in the coding or non-coding gene region. Those in the coding region may affect amino acid arrangement or influence protein kinetics, mRNA structure, and stability, while SNPs in the promoter region or other regulatory gene regions affect protein production (reviewed in 67, 78). According to the National Center for Biotechnology Information (NCBI) database on polymorphism, dbSNP (79), 24 polymorphisms in the MT2A gene have been identified in humans, three of which may affect physiological and pathophysiological processes. The most studied SNP in MT2A was rs28366003 (−5A/G), followed by rs10636 (+838G/C), whereas rs1610216 (−209A/G) has been the least investigated. Only Starska et al. (80, 81) and Krześlak et al. (82) studied all three MT2A SNPs and their associations with several malignant tumours in a Polish population.

Below we describe these and other information about MT2A polymorphisms with the focus on reported relationships with trace elements. We set up three sets of interrelated tables, in which we present the following groups of data for each SNP: 1) literature data on the related genotype frequencies; 2) reported associations with human diseases; and 3) reported associations with element concentrations in humans. Of the 36 selected references, 21 deal with relationships with elements (in healthy and/ diseased persons), 13 with diseases only, and two with genotype frequencies only.

There is strong evidence that MTs participate in physiological and pathological processes in the human body which involve the homeostasis of intracellular essential element, primarily Zn and Cu. They may chelate divalent toxic metals, such as Cd, Pb, Hg, or Pt with the –SH groups in cysteine and thus detoxify cells, scavenge free radicals, and protect cells against oxidative stress. They also have a role in cell survival and proliferation, angiogenesis, and inhibition of apoptosis. Emerging evidence confirms that MT insufficiency may lead to pathogenic processes and carcinogenesis. Single gene polymorphisms of MTs may be responsible for individual differences in reactions to harmful effects of external chemical and physical stressors and reactive oxygen species in the body.

Identification of individual MT isoforms in human cells and tissues can be applied in prospective tissue, plasma, and urine analyses or retrospectively, using fixed paraffin-embedded tissue samples. In the future, MTs may serve as biomarkers of environmental exposure to toxic metals, such as Cd, as already reported in biomonitoring studies on occupational exposure in humans (121, 122, 123) or environmental exposure in animals (124, 125, 126). MTs are also intensively studied as potential clinical biomarkers to be used in the diagnosis, prognosis, and selection of efficient therapy/ies for a number of malignant tumours, such as breast, thyroid, head, neck, lung, gallbladder, pancreas, colon, kidney, ovary, prostate, bone, and skin cancers, childhood solid tumours, and various types of leukaemia (29, 30, 31, 32, 82, 86, 87, 94, 127, 128, 129, 130, 131, 132). Exogenous MTs are already being investigated for the treatment of pathological processes in the central nervous system (59).

To date, the rs28366003, rs10636, and rs1610216 SNPs in the MT2A gene have been associated with various physiological and pathological conditions. These involve ageing and chronic diseases, such as metabolic syndrome (including type 2 diabetes mellitus and obesity), cardiovascular diseases, osteoporosis, and psychiatric disorders. They also seem to interfere with the effects of toxic drugs and pollutants. However, their use as risk predictors remains controversial. Identifying a single specific allelic variant associated with an individual trait, health or disease by gene-specific, candidate-driven studies (133) may fail to provide full information and risk assessment of certain diseases, which, as a rule, have polygenic origins (134, 135) and therefore need genome-wide association studies (136, 137). More comprehensive studies are needed to determine the role and potential for the clinical use of specific MT2A gene polymorphisms. These should recruit a large number of participants (several hundreds and more) with well-defined pathological process and take into account other factors and risks, such as specific environmental exposure and personal habits, genetic characteristics, and epigenetic makeup.

Polimorfizmi gena metalotioneina 2A u ljudi i njihova povezanost s bolestima i razinama elemenata u tragu