CHAPTER 1

Metal Toxicity – An Introduction

Robert R. Crichton*a
a Universite Catholique de Louvain, 1348 Louvain-la-Neuvre, Belgium. E-mail: Robert.crichton@uclouvain.be


Both essential and non-essential metal ions can cause toxicity. The essential metal ions for humans are the bulk metals sodium, potassium, calcium and magnesium and the trace metals manganese, iron, cobalt, copper, zinc and molybdenum, and, after outlining why they are required, we summarise the circumstances in which certain of them can be toxic. A number of non-essential metals, like cadmium, lead and mercury are not at all necessary for life but, when introduced into the human environment, they pose serious problems on account of their toxicity. Essential metal ion toxicity can be attributed to accumulation of excessive concentrations of the metal ion, often in specific tissues or organs. The toxicity of non-essential metal ions is a consequence of environmental exposure leading to their accumulation within the body, where they interfere with the functions of the essential metal ions. Both types of metal toxicity can, in principle, be treated by the use of appropriate metal ion chelators.


1.1 Introduction

Metal toxicity1 can be caused by both metal ions, which are considered to be essential for humans, like iron and copper, as well as by non-essential metals, like cadmium, lead and mercury, which are not at all necessary for life but which, when introduced into the human environment, can have toxic effects, often with disastrous consequences. So, we begin by asking what are the essential metal ions, why they are required, and under what circumstances certain of them can be toxic. We then discuss which are the non-essential metal ions which pose toxicity problems to the human population. For each of these two groups of metal ions, general chemical considerations and the basic principles involved in their toxicity are briefly considered, as well as how the metal ion is bound within cells or tissues, since this is a key element in devising strategies for its removal by chelation. Finally, we briefly review the sources and routes of exposure to metal toxicity, with particular reference to non-essential metals.

Whereas essential metal ion toxicity can be attributed to accumulation of excessive concentrations of the metal ion, often in specific tissues or organs, the toxicity of non-essential metal ions is a consequence of environmental exposure leading to their accumulation within the body. Both types of metal toxicity can, in principle, be treated by the use of appropriate metal ion chelators, and this constitutes the subject of this contribution to the RSC Metallobiology series.

1.2 Essential Metals

There are around twenty five elements that are required by most biological systems, including an important number of metal ions. However, for humans, there are ten essential metal ions. Of these, four: sodium, potassium, calcium and magnesium, can be considered as ‘bulk elements’, and together constitute approximately 99% of the metal ion content of the human body. The other five transition metals manganese, iron, cobalt, copper, zinc and molybdenum, are known as ‘trace elements’, with much lower dietary requirements than the bulk elements; they are nonetheless indispensable for human life.2

1.2.1 Sodium and Potassium

The two essential alkali metal ions Na+ and K+ (together with H+ and Cl) are only weakly bound to organic ligands and are extremely mobile. This makes them ideally suited to generate ionic gradients across biological membranes and to ensure the maintenance of osmotic balance, and this is precisely what they do. The Na+ and K+ contents of the average 70 kg adult human are about 100 g and 140 g, respectively, but their distribution in most mammalian cells is quite different. Na+, together with Cl, is the major electrolyte in the extracellular fluid, whereas K+ is retained within the cells. The concentration of Na+ in the extracellular fluid (i.e., the plasma) is maintained within narrow limits at about 145 mmol L−1, whereas the intracellular concentration is about 12 mmol L−1. In contrast, the concentration of K+ is 150 mmol L−1 within the cells and typically 4–5 mmol L−1 in the extracellular fluids. This concentration differential, which is maintained by the (Na+–K+)–ATPase of the plasma membrane, ensures a number of major biological processes, such as cellular osmotic balance, signal transduction and neurotransmission.

1.2.2 Magnesium and Calcium

In contrast to Na+ and K+, the alkaline earths Mg2+ and Ca2+ have intermediate binding strengths to organic ligands and are therefore less mobile. They play important structural and catalytic roles and, in the particular case of Ca2+, serve as a charge carrier and a trigger for signal transmission within the cell. Ca2+ is the fifth most abundant element and most abundant metal ion in the human body, representing 14% of body mass (1 kg for a 70 kg human), whereas Mg2+ accounts for only 19 mg. Most (99%) of the body’s Ca2+ is found within the biominerals that constitute bone and teeth, but the 1% found within cells and tissues has enormous importance in regulating a series of important cellular responses, from initial fertilisation to being a harbinger of cell death, intervening between these limits of the life of the cell to include secretion, mobility, metabolic control, synaptic regulation and gene regulation. Although Mg2+ represents the least abundant of the ‘bulk elements’, it is the most abundant cation within cells, with roughly half of cytosolic Mg2+ bound to ATP and most of the rest bound, together with K+, to ribosomes; the intracellular concentration of free Mg2+ is around 0.5 mM, and less than 0.5% of total body Mg2+ is in the plasma. Unlike the other three bulk cations, Mg2+ has a much slower water exchange rate, allowing it to play a structural role, for example, participating in ATP binding in many enzymes involved in phosphoryl transfer reactions—six of the ten reactions of glycolysis are phosphoryl transfers.

1.2.3 Zinc

Of the six trace metal ions, in senso stricto, zinc is not a transition metal ion. Zn has ligand binding constants intermediate between those of Mg2+ and Ca2+ and the other five transition metals, and, in marked contrast, does not have access to any other oxidation state other than Zn2+, which may be one of the reasons why it is found in more than 300 enzymes, representing all of the six classes of enzymes represented by the International Union of Biochemistry, since it avoids generation of free radicals. Zn2+ not only plays a structural and catalytic role, often functioning, like Mg2+, as a Lewis acid; it can also fulfil a very important function in the structural motifs known as ‘zinc fingers’ involved in the regulation of transcription and translation via its DNA- and RNA-binding. Zn2+ is the second most abundant of the trace metals (after iron), and is extensively involved in brain function. Bioinformatic analysis reveals that the zinc proteome represents about 10% of the entire proteome in humans, compared to 5–6% in prokaryotes.3

1.2.4 Other Essential Metals

The other five transition metal ions: Mn, Fe, Co, Cu and Mo, bind tightly to organic ligands, are therefore effectively immobile and participate in a wide range of redox reactions. Fe and Cu are constituents of a large number of proteins involved in electron transfer chains in humans, the respiratory chain in the inner membrane of the mitochondria, with Fe–S proteins, cytochromes and the terminal component, the Cu–Fe-dependent cytochrome c oxidase. Fe also plays an important role in the oxygen transport and storage proteins, haemoglobin and myoglobin, while both Fe and Cu are involved in oxygen activation (oxidases, hydroxylases) and detoxification (Cu–Zn superoxide dismutase). Co is required in the diet as vitamin B12, and is an essential cofactor for a number of B12-dependent isomerases and methyltransferases. Mn plays an important role in the detoxification of oxygen free radicals (mitochondrial Mn superoxide dismutase). Mo, while relatively rare in the earth’s crust, is the most abundant transition metal in seawater, and is an important component of nitrogenase, the key enzyme of nitrogen-fixing organisms. However, in humans there are a number of Mo-dependent enzymes, which all contain Mo in the form of a molybdenum pyranopteridin-dithiolate cofactor. They include xanthine oxidase, involved in the catabolism of purine bases, sulphite oxidase involved in sulphur metabolism and aldehyde oxidase, involved in the metabolism of many drugs.

Ni2+, V and Cr appear to be beneficial and have been proposed to be essential for man but we will not discuss their roles further.

1.3 Toxicity Due to Essential Metals

Alle Dinge sind Gift, und nichts ist ohne Gift; allein die dosis machts, daß ein Ding kein Gift sei. Paracelsus (1493–1541)

This celebrated dictum of Paracelsus, the Swiss physician, alchemist, mystic and philosopher (born Phillip von Hohenheim, later called Philippus Theophrastus Aureolus Bombastus von Hohenheim, and ultimately Paracelsus), can be loosely translated as ‘Everything is poisonous and nothing is not poisonous; only the dose determines whether something is poisonous or not’.

Nowhere is this more true than in describing the toxicity of essential metal ions. The concentration of each of the essential metal ions needs to be maintained within strict limits in each cell and tissue of the body. This is what the celebrated French physiologist Claude Bernard defined as homeostasis—’the fixity of the internal environment is the condition for free life’. He continued ″The living body, though it has need of the surrounding environment, is nevertheless relatively independent of it. This independence which the organism has of its external environment, derives from the fact that in the living being, the tissues are in fact withdrawn from direct external influences and are protected by a veritable internal environment which is constituted, in particular, by the fluids circulating in the body.″. Thus, any factor, whether hereditary, environmental or other, which perturbs the homeostatic equilibrium of a metal ion can result in a shift from that equilibrium state to a condition of either deficiency or excess, and it is the latter that is the origin of the toxicity of essential metals.

It is clearly beyond the scope of this chapter to discuss the homeostasis of each of the essential metal ions and in what follows we will highlight some of the major causes of essential metal toxicity in humans with a particular focus on those that are accessible to therapeutic treatment by chelation therapy and to identifying areas involving essential metal overload that may become amenable to chelation therapy.

1.3.1 Sodium and Potassium

Selectivity in the coordination of the alkali metals, Na+ and K+, can be easily achieved since the difference in their ionic radii is large (0.35 Å) and hence the dimensions of the cavity surrounding the unhydrated monovalent cation effectively solves the problem of Na+–K+ selectivity in both complexation and transport. This was established by early studies on synthetic model compounds,4 confirmed by the high resolution structures of membrane transporters of K+5,6 and, more recently, the structure of an Na+ transporter.7

The structure of the KcsA K+ channel5 from Streptomyces lividans, an integral membrane protein with sequence similarity to all known K+ channels, reveals that it is constructed in the form of an inverted teepee, with the narrow 12 Å long selectivity filter in its outer end. Whereas the central cavity of the pore is wider and lined with hydrophobic amino acids, the selectivity filter is lined by main chain carbonyl oxygen atoms (Figure 1.1a) and is held open by structural constraints to coordinate unhydrated K+ ions but not the smaller Na+ ions. Thus, the architecture of the pore establishes the physical principles underlying selective K+ conduction.5 In order to transport unhydrated K+ ions in the selectivity filter, the K+ ion’s hydration shell must be removed. The structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 Å resolution8 shows how this is achieved. The transfer of a K+ ion between the extracellular solution (where a K+ ion is hydrated) and the selectivity filter (where the ion is unhydrated) is mediated by a specific arrangement of carbonyl oxygen atoms that protrude into solution. The selectivity filter contains two K+ ions about 7.5 Å apart. When the configuration of ions (green spheres) and water (red spheres) inside the filter is K+–water–K+–water (top, 1,3 configuration), a K+ ion at the entryway is surrounded by eight ordered water molecules ready to enter the selectivity filter. When the configuration is water–K+–water–K+ (bottom, 2,4 configuration), the K+ ion at the filter threshold senses the electrostatic field due to the ion distribution within the filter and is drawn closer to the channel from the fully hydrated position to a position where it is half hydrated.

Fig. 1.1 (a) The KcsA K+ channel showing the selectivity pore and the K+ ion dehydration at the extracellular pore entryway. Reprinted by permission from Macmillan Publishers Ltd: Nature, Zhou et al.8 Copyright 2001. (b) A schematic depiction of the proposed mechanism of ion selectivity in NavAb. Reprinted with permission from Corry and Thomas.9 Copyright (2012) American Chemical Society.

Using the structure of the voltage gated sodium channel, NavAb from Arcobacter butzleri,7 the location and probable mechanism used to discriminate between Na+ and K+ has been pinpointed.9 The overall topography of the NavAb channel is similar to that of the K+ channel,8 in the form of an inverted tepee, but differs significantly from the K+ channel in the narrow ‘selectivity filter’, which is both wider and shorter as well as being lined by amino acid side chains. The proposed mechanism of ion selectivity in NavAb is presented in Figure 1.1b. The first surprise is that, whereas in the K+ channel5 (Figure 1.1b) unhydrated K+ ions transit the selectivity filter, hydrated Na+ ions pass through the NavAb channel. Although K+ could fit through the narrow portion of the pore with a complete hydration shell as shown in Figure 1.1b (A), it cannot do so due to the presence of charged glutamate residues Figure 1.1b (B). Selectivity arises due to the inability of K+ to fit between the plane of glutamate residues with the preferred solvation geometry, which involves water molecules bridging between the ion and carboxylate groups. In contrast, the smaller Na+ ion can fit in the pore with its preferred solvation geometry Figure 1.1b.

Retention of Na+ (hypernatraemia) is one of the most common electrolyte disorders in clinical medicine, occurring when Na+ intake exceeds renal clearance. It can be caused by excessive ingestion of salt, too-rapid infusion of saline, congestive heart failure, renal failure, or when there is excessive production of aldosterone, resulting in hypervolumaenia and hypertension.10 Hypernatraemia and dehydration are commonly encountered among the elderly, and when they occur in nursing homes are considered indicators of neglect.11 The treatment involves correcting the underlying cause and correcting the water deficit.

Hyperkalaemia,12,13 defined as serum K+ levels in excess of 5.5 mM, has become more common in cardiovascular practice due to the growing population of patients with chronic kidney disease and the broad application of drugs that modulate renal elimination of potassium by reducing production of angiotensin II (angiotensin-converting enzyme inhibitors, direct renin inhibitors, β-adrenergic receptor antagonists), blocking angiotensin II receptors (angiotensin receptor blockers), or antagonizing the action of aldosterone on mineral corticoid receptors (mineralocorticoid receptor antagonists). Current therapies for hyperkalaemia either do not remove excess potassium or have poor efficacy and tolerability. Two new polymer-based, non-systemic oral agents, patiromer calcium (RLY5016) and zirconium silicate (ZS-9), currently in development,12–14 are designed to induce potassium loss via the GI tract, particularly the colon, and reduce plasma K+ levels, and both have demonstrated efficacy and safety in recent trials. Patiromer sorbitex calcium is a polymer resin and sorbitol complex that binds potassium in exchange for calcium; ZS-9, a non-absorbed, highly selective inorganic cation exchanger, traps potassium in exchange for sodium and hydrogen.

Fig. 1.2 Catalytic cycle for Ca2+ conductance by CaVAb. Coupling of extracellular Ca2+ binding sites and the three sites within the selectivity filter in the two proposed ionic occupancy states. When two Ca2+ ions bind to position 1 and 3 in the filter, the entryway Ca2+ ion is placed furthest from the pore (left). When one Ca2+ ion binds to position 2 within the filter, the ion outside the filter is pulled closer to the pore (right). Reproduced by permission from Macmillan Publishers Ltd: Nature, Tang et al.15 Copyright 2013.

1.3.2 Calcium and Magnesium

Differences in Ca2+versus Mg2+ complexation are more difficult to achieve than between K+ and Na+, although the recent structure determinations of Ca2+ and Mg2+ channels promises to improve our understanding of how selectivity is achieved.15,16 The selectivity filter of a voltage-gated Ca2+ channel is presented in Figure 1.2.

Calcium and, to a lesser extent, magnesium balance is achieved through a complex interplay between the parathyroid gland, bone, the intestine and the kidney.17,18 Hypercalcaemia is a common metabolic perturbation and the most common causes are malignancy and hyperparathyroidism, which account for around 80% of cases,17 although the increase in over-the-counter purchase of Ca2+ and vitamin D supplements, notably to combat osteoporosis in the ageing population, is also a factor.19,20 Management of hypercalcaemia has been based on the use of bisphosphonates (e.g., zoledronic acid) and calcimimetic agents, although recently the nuclear factor-κ ligand monoclonal antibody Denosumab has also been introduced for the treatment of hypercalcaemia of malignancy.21,22 Clinically significant hypermagnesaemia is uncommon, generally occurring only in the setting of renal insufficiency and excessive magnesium intake.

1.3.3 Zinc

The essential transition metal ions, manganese, iron, cobalt, copper and zinc present a very different picture concerning their complexation. It is difficult to selectively chelate the divalent state of these metal ions, which have very similar coordination preferences. However the selective complexation of Fe3+ and Cu+ is relatively easy. Fe3+ has no other biological counterpart in terms of its preference for hexacoordinate geometry with predominantly ‘hard’ ligands such as oxygen, although, as we will see later, Al3+ and Ga3+ can use the iron transport protein transferrin as Fe3+ surrogates. In marked contrast, the rather ‘soft’ Cu+ ion is ideally coordinated by sulphur ligands, and it comes as no great surprise that this is the coordination used by intracellular copper chaperones.

The human body contains about 2 g of Zn, mostly found in testes, muscle, liver, and brain. Zinc deficiency due, for example, to poor nutrition, ageing, and deregulation of zinc homeostasis, is much more frequently encountered in the human population than zinc excess.23 Only exposure to high doses has toxic effects. However, long-term, high-dose zinc supplementation interferes with copper uptake, such that many of the toxic effects are a consequence of copper deficiency. Zinc-induced myeloneuropathy resulting from secondary copper deficiency has been recently rediscovered;24,25 in reports published by Schlockow dating from the 1870s24 this was a recognised problem among zinc smelter workers in Upper Silesia who developed symptoms identical to those reported in the modern descriptions of copper-deficiency myeloneuropathy.24,25 The highest concentration of brain zinc is found in the hippocampus, amygdala, cerebral cortex, thalamus, and olfactory cortex.26 Some 10% of brain zinc is histochemically detectable by chelating agents, mainly stored in the presynaptic vesicles of specific excitatory glutamatergic neurons, and is secreted from these vesicles into the synaptic clefts along with glutamate during neuronal excitation. Although the role of Zn in the brain remains elusive, recent studies suggest that secreted Zn2+ plays crucial roles in information processing, synaptic plasticity, learning, and memory.27,28 There is considerable evidence that either deficiency or overload of Zn in the brain, resulting from perturbations in Zn homeostasis, are associated with the pathogenesis of several neurodegenerative diseases including senile dementia (Alzheimer’s disease (AD) and vascular dementia),29–35 prion diseases,36 and amyotrophic lateral sclerosis (ALS).37 It has been proposed that Zn2+ may play two roles35 in the pathogenesis of AD. On the negative side, in common with other metals, Zn2+ enhances the oligomerization of amyloid beta (Aβ) peptide, which is deposited in the neurotic plaques that characterise AD, yet it also appears to have a protective function by inhibiting Aβ peptide-induced Ca2+ entry into amyloid channels, which disrupts Ca homeostasis and provokes neuronal death.

1.3.4 Other Essential Metals

1.3.4.1 Cobalt Toxicity

Cobalt is an essential micronutrient in the form of vitamin B12 (hydroxocobalamin), present as a corrin cofactor in several enzymes, but inorganic cobalt is not required as such in human diets. Methionine aminopeptidase (MetAP), which removes the initiator methionine residue from the N-terminus of nascent polypeptide chains, and is conserved from yeast to humans, is a candidate to be a non-corrin Co2+-activated enzyme in humans.38 The MetAPs, essential from bacteria to higher eukaryotes, are dinuclear metalloenzymes with evolutionary similarities to creatinase, prolidase (DPP) and aminopeptidase P (APP).39 Metal requirement studies indicate that MetAPs can be activated by various divalent metal cations.40

Cobalt is acutely toxic in large doses41,42 and the consequences were dramatically observed in the 1960s among heavy beer drinkers (15–30 pints per day), when Co2+ salts were added to beer as foam stabilisers,43,44 resulting in severe and often lethal cardiomyopathy. The effects were virtually absent in well nourished drinkers whereas an identical Co dose was severe and often resulted in death.45 Evidence of Co toxicity was also found when Co was used therapeutically to treat anaemia.41 However, since these practices have been discontinued, several subpopulations with elevated Co exposures have emerged, confirming that cumulative, long-term exposure, even at a low level, can give rise to adverse health effects related to various organs and tissues. These include occupational cobalt exposure, consuming Co-containing dietary supplements, the misuse of Co as a blood doping agent by athletes (Co stabilises hypoxia-inducible factor, mimicking hypoxia and stimulating erythropoiesis) and most recently, concerns about elevated blood Co levels in patients who have undergone orthopaedic joint replacements with cobalt–chromium hard metal alloys.41,42 Corrosion and wear produce soluble metal ions and debris in the form of Co–Cr nanoparticles, from which Co ions are released, and so we progress from nanotechnology to nanotoxicology. It is suggested that implant patients should be monitored for signs of hypothyroidism and polycythaemia when levels of Co in the circulation exceed 100 µg L−1.42

1.3.4.2 Manganese Toxicity

Overexposure to manganese leads to toxicity, particularly neurotoxicity.46–52 Neurons are more susceptible than other cells to Mn-induced toxicity, and accumulation of Mn in the brain results in the condition known as manganism, first observed in miners during the 19th century, which presents with Parkinson’s disease-like symptoms. Mn neurotoxicity has been attributed to impaired dopaminergic, glutamatergic and GABAergic neurotransmission, disruption of mitochondrial function leading to oxidative stress and neuroinflammation. Preferential accumulation of Mn in dopaminergic cells of the basal ganglia, in particular the globus pallidus, results in the extrapyrimidal motor dysfunction characteristic of manganism. Causes of Mn toxicity include occupational and environmental exposures as well as mutations in the SLC30A10 gene, recently identified as a Mn transporter in humans. Current treatment strategies combine chelation therapy and iron supplementation, the latter to reduce Mn binding to proteins that interact with both Mn and Fe.

1.3.4.3 Iron and Copper Toxicity

Both iron and copper are characterised by genetic disorders associated with accumulation of the metal in particular tissues, resulting in toxic consequences. There are two classic disorders of Cu metabolism, which are both caused by defective copper transporting ATPases.53–55 Menkes disease is an X-chromosome linked fatal neurodegenerative disorder of childhood characterised by massive copper deficiency.56 In contrast, Wilson’s disease (WD) is a chronic disease of brain and liver, accompanied by progressive neurological dysfunction, due to a disturbance of copper metabolism, with progressive accumulation of copper in the brain, liver, kidneys, and the cornea of the eye.

WD is caused by mutations in the ATP-driven copper transporter ATP7B,57 which is expressed in various tissues, including liver and the central nervous system.58 In the hepatocyte, copper is taken up via CTR1 (Figure 1.3), and the Cu+ is delivered by the copper chaperone ATOX1 to ATP7B.59 ATP7B then transports copper from the cytosol into the lumen of the trans-Golgi network (TGN) for incorporation into secreted copper-dependent enzymes, such as ceruloplasmin (Figure 1.4). In conditions of copper loading, it also transports copper into vesicles for export into bile.60 This biliary excretion process involves another protein, COMMD1 (originally called MURR1), which interacts directly with ATP7B.61

Fig. 1.3 Pathways of copper metabolism in the hepatocyte. Cu – copper. CTR1 – copper transporter 1. MT – metallothioneins. GSH – glutathione. Cp – ceruloplasmin. Reproduced from The Lancet, 369: 9559, Aftab Ala et al. Wilson’s Disease, 397–408, Copyright 2007 with permission from Elsevier.
Fig. 1.4 Regulation of hepatic hepcidin production. Transferrin-iron is a critical indicator for systemic iron homeostasis and a regulator of hepcidin expression. Reproduced from Steinbicker and Muckenthaler, 2013.64 This is an open access article distributed under the Creative Commons Attribution License.

In WD, ATP7B expression, function, and/or intracellular targeting are disrupted by mutations, resulting in impairment of both copper delivery to the trans Golgi network and copper excretion, causing copper to accumulate to very high levels in the liver. WD can be effectively treated, the aim of treatment is to reduce the amount of free copper. WD therapy has not really evolved over the last two decades and relies essentially on chelation therapy with copper chelators (penicillamine, trientine, and tetrathiomolybdate), zinc salts, or both.55 Chelating agents bind copper directly in blood and tissues and facilitate its excretion, whereas zinc interferes with the intestinal uptake of copper. Early recognition of the disease by means of clinical, biochemical or genetic examination and initiation of therapy is essential for a favourable outcome. For WD patients who present with acute liver failure or end-stage cirrhosis, liver transplantation is the only effective treatment, see Chapter 4.

Iron overload is essentially due to genetic defects in iron absorption from the gastrintestinal tract.62,63 Systemic iron homeostatic balance is regulated by hepcidin, a peptide released by the liver that binds to the iron exporter ferroportin, blocking the export of iron from intestinal cells, hepatocytes and macrophages when iron is in excess, and permitting its export from these same cells when iron is in short supply. Hepatic hepcidin synthesis is regulated by iron, bone morphogenetic protein signaling, inflammation, erythropoiesis, hypoxia, or endocrine stimuli (Figure 1.4). FPN1, which is expressed predominantly in hepatocytes, macrophages and enterocytes is internalized and degraded following hepcidin binding.

Thus, there will be two major classes of disease resulting from disequilibrium of hepcidin synthesis: anaemia and iron loading. Elevated levels of hepcidin will decrease ferroportin expression, trap iron within enterocytes, hepatocytes and macrophages, and decrease gut iron absorption resulting in iron deficiency. In contrast, inappropriately low levels of hepcidin will result in uncontrolled expression of ferroportin, increasing iron absorption from the gut and its release from hepatocytes and macrophages, causing iron overload. This is what we see in hereditary haemochromatosis (HH), which will not be considered here since its therapy involves venesection, but also in secondary haemochromatosis resulting from genetic dysfunction of erythropoiesis, which clearly requires chelation therapy and is described in Chapter 4.

1.3.4.4 Molybdeum Toxicity

The last of the essential transition metal ions, molybdenum, is present within the pterin-based molybdenum cofactor (Moco) in the active site of four mammalian enzymes and, while there is a large literature concerning the frequently fatal consequences of Mo deficiency, cases of Mo toxicity are much rarer.64 Epidemiological studies suggest that living near mountaintop coal mining activities is one of the contributing factors for high lung cancer incidence. A recent study established the long-term carcinogenic potential of molybdenum, the main inorganic chemical constituent of MTM particulate matter.65

1.4 Non-Essential Metals

The toxicity of heavy metals is one of the oldest environmental problems and remains a serious health concern today. But what exactly are these heavy metals? Consulting any database for ‘heavy metal’ one might rapidly conclude that it concerns the development of a particular form of hard rock music characterised by a massive, highly amplified, distorted and very loud sound with aggressive male chauvinist lyrics. Alternatively, heavy metals used to be defined as dense metals or metalloids, which were potentially toxic, notably in an environmental context—typical heavy metals are cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As). We prefer to designate the ‘heavy metals’ as non-essential toxic metals, and we will devote most of our attention to Cd and Pb, Hg and As, although we will encounter a few others along the way. These will include chromium, thalium, and, why not, aluminium, implicated in Alzheimer’s disease—the curse of our ageing population. Then again, there is the silent menace posed by radionuclides, when something as disastrous as Chernobyl, Three Mile Island or Fukoshima occurs, not forgetting polonium-210, hitting the headlines very publicly when it was used to poison the Russian dissident Alexander Litvinchenko in London in a very English cup of tea. Their selective chelation is dealt with in Chapter 6.

We will not consider here non-essential metal ions that are employed in therapy, such as Pt and Ru derivatives in cancer treatment, Li for manic depression or Au for rheumatoid arthritis, nor those such as Gd derivatives used as MRI contrast agents—their potential toxicity is the price we are prepared to pay for their therapeutic or diagnostic value.

1.5 Toxicity Due to Non-Essential Metals

1.5.1 Lead

Thirty years ago, Jerome Nriagu argued in a paper,66 which was widely discussed in the press at the time, that Roman civilization collapsed as a result of chronic lead poisoning (saturnism). We are now acutely aware that saturnism is a major cause of environmental concern, although the Roman world was clearly unaware of these risks. However, whereas lead is no longer seen as the prime culprit of the Fall of Rome, a recent study67 has shown that, nonetheless, “tap water” from ancient Rome had 100 times more Pb than local spring waters. Pb toxicity affects several organ systems including the nervous, haematopoietic, renal, endocrine and skeletal systems. It causes behavioural and cognitive deficits during brain development in infants and young children, and there is growing evidence that exposure to Pb in early life may predispose to neurodegeneration later in life. Despite numerous global initiatives to reduce the use of Pb, Pb exposure remains a widespread problem,68,69 particularly in the developing world. Even in the last decade, blood lead levels (BLL) in children living in Pb polluted areas of China, India and Egypt have exceeded the 10 µg dL−1 level set in 1991, and subsequently reduced to zero by the US Disease Control and Prevention Centre, in 10–44% of tested children.70

Pb appears to target proteins that naturally bind calcium and zinc.71 Examples of proteins that are targeted by lead include synaptotagmin, which acts as a calcium sensor in neurotransmission, and δ-aminolaevulinate synthase (ALAD), the second enzyme in the haem biosynthetic pathway (Figure 1.5). Despite its size, lead (1.19 Å) can substitute for calcium (0.99 Å) in synaptotagmin and zinc (0.74 Å) in ALAD. Human ALAD is activated by Zn2+ with a Km of 1.6 pM and inhibited by Pb2+ with a Ki of 0.07 pM. Pb2+ and Zn2+ appear to compete for a single metal binding site.72 Like Mn, Pb causes presynaptic dysfunction but, in contrast to Mn, which affects the dopaminergic system, Pb2+ appears to interfere with hippocampal glutamatergic neurotransmission.73

Fig. 1.5 Lead can substitute for calcium in synaptotagmin and zinc in δ-aminolaevulinate synthase (ALAD). Reproduced from Current Opinion in Chemical Biology, 5, 2, Hilary Arnold Godwin, The biological chemistry of lead, 223–227, Copyright 2001 with permission from Elsevier.

1.5.2 Cadmium

Cd2+, a soft Lewis acid with a preference for easily oxidized soft ligands, particularly sulphur, can displace Zn2+ from proteins where the Zn coordination environment is sulphur dominated, while, on account of the similarity of its ionic radius with that of Ca2+, it can exchange with Ca2+ in calcium-binding proteins.74 Both Zn2+ and Cd2+ induce the iron export protein ferroportin (FPN1) transcription through the action of Metal Transcription Factor-1 (MTF-1)75 and it has recently been shown that both Zn2+ and Cd2+ are transported by FPN1.76 Cd2+ bioavailability and retention is favoured by poor iron status.

Cadmium occurs in the environment naturally and as a pollutant emanating from industrial and agricultural sources. Exposure to cadmium in the non-smoking population occurs primarily through food, and chronic exposure results in respiratory disease, emphysema, renal failure, bone disorders and immuno-suppression.77 Recent data also suggest increased cancer risks and increased mortality in environmentally exposed populations.78 At the cellular level, Cd2+ affects proliferation, differentiation and causes apoptosis. However, since Cd2+ is not redox active, the generation of reactive oxygen species (ROS) and DNA damage must be due to indirect effects. Recent studies indicate that Cd is able to induce various epigenetic changes,79,80 such as DNA methylation, histone modification, and non-coding RNA expression. It also modulates gene expression and signal transduction, reduces the activities of proteins involved in antioxidant defences, and interferes with DNA repair, see Chapter 3.

The most commonly used therapeutic strategy for heavy metal poisoning is chelation therapy to promote metal excretion. Chelators such as CaNa2EDTA and meso-2,3-dimercaptosuccinic acid (DMSA) have been reported to have protective effects against Cd toxicity.81,82

1.5.3 Mercury

The brutal reality of mercury toxicity was highlighted in 1956 by an environmental disaster that struck the population of Minamata, Japan and its surroundings; we now refer to mercury toxicity as Minamata disease.83 Of the 2265 victims officially recognised by March 2001, 1784 died. This neurological syndrome is caused by severe mercury poisoning with symptoms including ataxia, numbness in the hands and feet, general muscle weakness, narrowing of the field of vision and damage to hearing and speech. As we point out later, it was only in 2004 that the chemical company responsible for the pollution was obliged to clean up its contamination.

Of the three biological forms of Hg: organic mercury (predominantly methyl mercury, MeHg), metallic mercury, and inorganic mercury compounds (principally mercuric chloride), MeHg was the one responsible for the Minamata hecatomb. MeHg is derived from the methylation of inorganic mercury in aquatic sediments and soils, is well absorbed from the diet and distributes within a few days to all tissues of the body. It is present as water-soluble complexes attached to thiol ligands, and crosses the blood–brain barrier as an MeHg–cysteine complex. The brain is the principal target tissue of MeHg and its major toxic effects are on the central nervous system, accumulating particularly in astrocytes. While it might be expected that the biochemical targets of Hg would be thiol groups of SH-dependent enzymes, it turns out that selenocysteine has a much higher affinity for Hg than cysteine. In reality, while Se can act as an effective blocker of Hg toxicity, the biological effects of Hg are much more direct.84 Selenoenzymes, in particular glutathione peroxidise, thioredoxin reductase and thioredoxin glutathione reductase, are required to prevent and reverse oxidative damage in the brain and neuroendocrine system,85 and these enzymes themselves are vulnerable to irreversible inhibition by methyl mercury (MeHg).86 Selenoenzyme inhibition appears likely to cause most if not all of the pathological effects of mercury toxicity, as outlined in Figure 1.6. A simplified portrayal of the normal cycle of selenoprotein synthesis is depicted on the left. Disruption of this cycle by exposure to toxic quantities of Hg (MeHg) is depicted on the right. Selenide freed during selenoprotein breakdown becomes bound to Hg, forming HgSe that accumulates in cellular lysosomes. If Hg is present in stoichiometric excess, formation of insoluble Hg selenides abolishes the bioavailability of Se for protein synthesis and results in loss of normal physiological functions that require selenoenzyme activities, see Chapter 3.86

Fig. 1.6 Schematic of Se sequestration mechanism of Hg toxicity. A simplified portrayal of the normal cycle of selenoprotein synthesis is depicted on the left. Disruption of this cycle by exposure to toxic quantities of Hg (MeHg) is depicted on the right. Reproduced from Toxicology, 278, N. V. C. Ralston and L. J. Raymond, Dietary selenium’s protective effects against methylmercury toxicity, 112–123, Copyright 2010 with permission from Elsevier.

1.5.4 Aluminium

Al3+, although the most abundant metal in the Earth’s crust, has been excluded from biological systems, essentially due to its lack of bioavailability. However, as we discuss later, several factors have increased the access of Al to the biosphere.87,88 This enhanced bioavailability has resulted in the accumulation of the metal in living organisms including humans, particularly in the skeletal system, the liver, and the brain. The toxicity of Al3+ is associated with anaemia, osteomalacia, hepatic disorders, and certain neurological disorders, notably Alzheimer’s disease.89 These disorders can be the consequence of long term chronic exposure to the metal, for example, in dialysis patients where Al3+ has been used as a phosphate buffer.90

The molecular targets of Al toxicity involve disruption of the homeostasis of essential metal ions, notably Fe, Ca and Mg.91,92 Al can replace Ca in the bone and interfere with Ca-based signalling events, while Mg binding to phosphate groups on cell membranes, ATP, and DNA can be replaced by Al. However, it is likely that the main targets of Al toxicity are Fe-dependent biological processes. As we pointed out earlier, Al3+ and Ga3+ both have coordination geometry similar to Fe3+, which, in principle, should enable Al3+ to subvert the plasma iron transport pathway. Although Al3+ has a lower affinity for transferrin (Tf) than Fe3+, Al3+ can indeed bind to transferrin,93 as illustrated in Figure 1.7 in the structure of Al3+–transferrin.94 However, no interaction between TfAl2 and TfR is detectable in in vitro binding studies.95 In the cytosol, Al3+ is unlikely to be incorporated into ferritin, which requires redox cycling between Fe2+ and Fe3+. It seems likely that most aluminium accumulates in mitochondria, where it can interfere with Ca2+ homeostasis.

Fig. 1.7 Aluminum-bound ovotransferrin (PDB: 2D3I) and iron-bound ovotransferrin (PDB: 1OVT) are shown in gray and orange, respectively. Aluminium and iron are shown as gray and orange spheres, respectively. Metal binding sites and bound carbonate anion of both forms are shown as stick models. Reproduced from Biochimica et Biophysica Acta (BBA) – General Subjects, 1820, K. Mizutani et al., X-ray structures of transferrins and related proteins, 203–211, Copyright 2012 with permission from Elsevier.

Regardless of the pathway by which it gains access to cells, Al3+ exerts many of its toxic effects by interfering with iron homeostasis, generating oxidative stress (Figure 1.8). Free Fe within the cell produces ROS through Fenton chemistry, which then interfere with numerous cellular constituents including lipids, protein, and nucleic acids.

Fig. 1.8 Al toxicity leads to defective Fe homeostasis and oxidative stress. Reproduced from Journal of Inorganic Biochemistry, 105, J. Lemire and V. D. Appanna, Aluminium toxicity and astrocyte dysfunction: A metabolic link to neurological disorders, 1513–1517, Copyright 2011 with permission from Elsevier.

1.6 Sources and Routes of Exposure with Particular Reference to Non-Essential Metals

Sources of low-level environmental exposure to Pb, in addition to water from lead pipes, include lead-based paint and household dust from surfaces covered with such paints, and Pb in air and food. The main routes for Pb are inhalation and ingestion, with inhalation being the more efficient route of absorption. Since Pb can adsorb onto particulate matter and thus be inhaled, the removal of the Pb4+-derived anti-knock agent (tetraethyl lead), which was commonly added to petrol to improve automotive engine efficiency, has greatly reduced blood Pb levels in the urban population.

Minamata disease was caused by the release of methyl mercury in the industrial wastewater from a chemical factory, where it bioaccumulated in aquatic food chains reaching its highest concentrations in shellfish and fish which, when eaten by the local population, resulted in mercury poisoning. In 1956 symptoms such as sensory disturbance in the distal portions of all extremities, partial paralysis, cerebellar ataxia, bilateral concentric contraction of the visual field, disturbed ocular movement, impairment of hearing and equilibrium disturbance were exhibited in increasing numbers of persons, including newborns. The disease, later described as “Minamata disease”, affected mainly local fishermen and their families. Fish-eating animals in the area (cats and seagulls) had similar neurological signs and led to the identification of an obvious heavy metal intoxication. However, it took several years to show the connection to a chemical factory that, for 36 years until 1968, discarded about 30 t of methyl mercury-associated waste into the Minamata Bay. MeHg present in fish and shellfish from the Bay was consumed in the surrounding area led to the various symptoms. As of March 2001, 2665 victims had been officially recognised as having Minamata disease (1784 of whom have died).

The second severe epidemic of methyl mercury poisoning happened in Iraq in 1972 and arose from the consumption of mercury-contaminated bread. This was the result of baking bread from wheat seed that had been treated with a mercury-based fungicide and was not supposed to have been planted.86 As a result, about 7000 people were affected and 460 died from mercury poisoning. Both the Japanese and Iraq methyl mercury poisoning incidents produced not only deaths, but also multiple and long-lasting symptoms, mainly in children and newborns, that included blindness, deafness, mental retardation, and cerebral palsy.

Asia is today the largest contributor of anthropogenic atmospheric mercury (Hg), accounting for over half of global emissions, with serious Hg pollutions to the local environment influenced principally by the chemical industry and by mining of gold and mercury. Studies have shown that in humans and selected Arctic marine mammals and birds of prey there has been and order of magnitude increase in Hg that began in the mid to late 19th century and accelerated in the 20th century. The man-made contribution to present day Hg concentrations is estimated at 92%.

There are several factors that account for the increased access of Al3+ to the biosphere. Firstly, an increase in anthropogenic acidification of soils, due to acid rain generated by emissions of sulphur dioxide and nitrogen oxides in the atmosphere, has resulted in elevated concentrations of Al3+ in ground waters. On account of its lightness and corrosion resistance, aluminium is widely used for industrial purposes, from the aerospace industry to construction, from food packaging to pharmaceuticals. Aluminium salts are extensively utilised as a flocculent in water treatment.

Cd2+ is a widespread environmental contaminant, with exposure largely via the respiratory or gastrointestinal tracts. The most important non-industrial sources of exposure are cigarette smoke and contaminated food and beverages. Cd2+ has a high rate of transfer from soil to plants, and certain plant species, including tobacco, rice, wheat, peanuts and cocoa accumulate large amounts of Cd2+ even from soils with a low Cd2+ content. In Europe, the highest levels of Cd2+ were found in topsoil soon after the distribution of P2O5, suggesting that the soil contamination is derived from the use of rock phosphate fertiliser in intensive arable agriculture. The effects of chronic oral ingestion of Cd2+ first manifested themselves in the form of Itai–itai disease among the inhabitants of the Jinzu river basin in Toyama Prefecture, Japan. This was the first time that Cd2+ pollution was shown to have severe consequences for human health, particularly in women. The most important effects were softening of the bones and kidney failure, the name of the disease deriving from the painful screams caused by the excruciating pain in the joints and the spines of the victims. The cause of the disease was traced to environmental pollution originating in effluent from a zinc mine located in the upper reaches of the river (Figure 1.9). In the case of Itai–itai disease most of the Cd2+ ingested orally was derived from contaminated rice.

Fig. 1.9 Itai–itai disease. Left: the degree of Cd pollution in parts of the endemic area; right: prevalence of the disease in women over 50 years of age.

1.7 Conclusions

In this brief overview we have outlined the importance of metal toxicity involving both essential and non-essential metal ions. It is clear that excessive accumulation of either of these classes of metal ion can result in a variety of toxic effects. The objective of this volume in the RSC Metallobiology series is to provide a clear and up-to-date perspective on the therapeutic potential of chelating agents in the management of metal excess. Such chelating agents should, as far as possible, fulfil the following criteria: (i) display a degree of selectivity towards the metal ions that they are intended to remove (for example, Fe3+ can be chelated selectively relatively easily from other essential transition metal ions); (ii) access potential sites where chelation is desired; (iii) cause minimal, if possible zero, interference with essential biologically important metal ions and the metabolic processes in which they are involved; (iv) be excreted from the cellular site of chelation and subsequently eliminated from the body without causing toxic effects en route (e.g., nephrotoxicity).

Abbreviations

AD
Alzheimer’s disease
ALS
Amyotrophic lateral sclerosis
BLL
Blood lead levels
HH
Hereditary haemochromatosis
MR (MRI)
Magnetic resonance (imaging)
ROS
Reactive oxygen species
WD
Wilson’s disease

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