## 4.1.3. Tissue accumulation of metals

Author: Nico M. van Straalen

Reviewers: Philip S. Rainbow, Henk Schat

Learning objectives:

You should be able to

- indicate four types of inorganic metal binding cellular constituents present in biological tissues and indicate which metals they bind.
- describe how phytochelatin and metallothionein are induced by metals.
- mention a number of organ-metal combinations that are critical to metal toxicity.

Key words: Metal binding proteins; phytochelatin; metallothionein;

Synopsis

The issue of metal speciation, which is crucially important to understand metal fate in the environment, is equally important for internal distribution in organisms and toxicity inside the cell. Many metals tend to accumulate in specific organs, for example the hepatopancreas of crustaceans, the chloragogen tissue of annelids, and the kidney of mammals. In addition, there is often a specific organ or tissue where dysfunction or toxicity is first observed, e.g., in the human body, the primary effects of chronic exposure to mercury are seen in the brain, for lead in bone marrow and for cadmium in the kidney. This module is aimed at increasing the insight into the different mechanisms by which metals accumulate in biological tissues.

Introduction

Metals will be present in biological tissues in a large variety of chemical forms: free metal ion, various inorganic species with widely varying solubility, such as chlorides or carbonates, plus all kind of metal species bound to low-molecular and high-molecular weight biotic ligands. The free metal ion is considered the most relevant species relating to toxicity.

To explain the affinities of metals with specific targets, a system has been proposed based on the physical properties of the ion; according to this system, metals are divided into “oxygen-seeking metals” (class A, e.g. lithium, beryllium, calcium and lanthanum), and “sulfur-seeking metals” (class B, e.g. silver, mercury and lead) (See section on Metals and metalloids). However, most of the metals of environmental relevance fall in an intermediate class, called “borderline” (chromium, cadmium, copper, zinc, etc.). This classification is to some extent predictive of the binding of metals to specific cellular targets, such as SH-groups in proteins, nitrogen in histidine or carbonates in bone tissue.

Not only do metals differ enormously in their physicochemical properties, also the organisms themselves differ widely in the way they deal with metals. The type of ligand to which a metal is bound, and how this ligand is transported or stored in the body, determines to a great extent where the metal will accumulate and cause toxicity. Sensitive targets or critical biochemical processes differ between species and this may also lead to differential toxicity.

Inorganic metal binding

Many tissues contain “mineral concretions”, that is, granules with a specific mineral composition that, due to the nature of the mineral, attract different metals. Especially the gut epithelium of invertebrates, and their digestive glands (hepatopancreas, midgut gland, chloragogen tissue) may be full of such concretions. Four classes of granules are distinguished (Figure 1):

- Calcium-pyrophosphate granules with magnesium, manganese, often also zinc, cadmium, lead and iron
- Sulfur granules with copper, sometimes also cadmium
- Iron granules, containing exclusively iron
- Calcium carbonate granules, containing mostly calcium

Figure 1. Schematic diagram of the gut epithelium of an invertebrate animal, showing four different types of granules and the metals they usually contain. Redrawn from Hopkin (1989) by Wilma IJzerman.

The type B granules are assumed to be lysosomal vesicles that have absorbed metal-loaded peptides such as metallothionein or phytochelatin, and have developed into inorganic granules by degrading almost all organic material; the high sulfur content derives from the cysteine residues in the peptides.

Tissues or cells that specialize in the synthesis of intracellular granules are also the places where metals tend to accumulate. Well-known are the “S cells” in the hepatopancreas of isopods. These cells (small cells, B-type cells sensu Hopkin 1989) contain very large amounts of copper. Most likely the large stores of copper in woodlice and other crustaceans relate to their use of hemocyanin, a copper-dependent protein, as an oxygen-transporting molecule. Similar tissues with high loadings of mineral concretions have been described for earthworms, snails, collembolans and insects.

Organic metal binding

The second class of metal-binding ligands is of organic nature. Many plants but also several animals synthesize a peptide called phytochelatin (PC). This is an oligomer derived from glutathione with the three amino acids, γ-glumatic acid, cysteine and glycine, arranged in the following way: (γ-glu-cys)n-gly, where n can vary from 2 to 11. The thiol groups of several cysteine residues are involved in metal binding.

The other main organic ligand for metals is metallothionein (MT). This is a low-molecular weight protein with hydrophilic properties and an unusually large number of cysteine residues. Several cysteines (usually nine or ten) can bind a number of metal ions (e.g. four or five) in one cluster. There are two such clusters in the vertebrate metallothionein. Metallothioneins occur throughout the tree of life, from bacteria to mammals, but the amino acid sequence, domain structure and metal affinities vary enormously and it is doubtful whether they represent a single evolutionary-homologous group.

In addition to these two specific classes of organic ligands, MT and PC, metals will also bind aspecifically to all kind of cellular constituents, such as cell wall components, albumen in the blood, etc. Often this represents the largest store of metals; such aspecific binding sites will constantly deliver free metal ions to the cellular pool and so are the most important cause of toxicity. Of course metals are also present in molecules with specific metal-dependent functions, such as iron in hemoglobin, copper in hemocyanin, zinc in carbonic anhydrase, etc.

The distinction between inorganic ligands and organic ones is not as strict as it may seem. After binding to metallothionein or phytochelatin metals may be transferred to a more permanent storage compartment, such as the intracellular granules mentioned above, or they may be excreted.

Regulation of metal binding

Free metal ions are strong inducers of stress response pathways. This can be due to the metal ion itself but more often the stress response is triggered by a metal-induced disturbance of the redox state, i.e. an induction of oxidative stress. The stress response often involves the synthesis of metal-binding ligands such as phytochelatin and metallothionein. Because this removes metal ions from the active pool it is also called metal scavenging.

The binding capacity of phytochelatin is enhanced by activation of the enzyme phytochelatin synthase (PC synthase). According to one model of its action, the C-terminus of the enzyme has a “metal sensor” consisting of a number of cysteines with free SH-groups. Any metal ions reacting with this nucleophile center (and cadmium is a strong reactant) will activate the enzyme which then catalyzes the reaction from (γ-glu-cys)n-gly to (γ-glu-cys)n+1-gly, thus increasing the binding capacity of cellular phytochelatin (Figure 2). This reaction of course relies on the presence of sufficient glutathione in the cell. In plants the PC-metal complex is transported into the central vacuole, where it can be stabilized through incorporation of acid-labile sulfur (S2). The PC moiety is degraded, resulting in the formation of inorganic metal-sulfide crystallites. Alternatively, complexes of metals with organic acids may be formed (e.g. citrates or oxalates). The fate of metal-loaded PC in animal cells is not known, but it might be absorbed in the lysosomal system to form B-type granules (see above).

Figure 2. Model for the regulation of phytochelatin synthase, an enzyme catalyzing the extension of phytochelatin and increasing the binding capacity for metals. From Cobbet (2000). Source: http://www.plantphysiol.org/content/123/3/825

The upregulation of metallothionein (MT) occurs in a quite different manner, since it depends on de novo synthesis of the apoprotein. It is a classic example of gene regulation contributing to protection of the cell. In a wide variety of animals, including vertebrates and invertebrates, metallothionein genes (Mt) are activated by a transcription factor called metal-responsive transcription factor 1 (MTF-1). MTF -1 binds to so-called metal-responsive elements (MREs) in the promoter of Mt. MREs are short motives with a characteristic base-pair sequence that form the core of a transcription factor binding site in the DNA. Under normal physiological conditions MTF-1 is inactive and unable to induce Mt. However, it may be activated by Zn2+ ions, which are released, from unspecified ligands, by metals such as cadmium that can replace zinc (Figure 3).

Figure 3. Model for metallothionein induction by cadmium. Cadmium ions (Me) displace zinc ions from ligands (MP). Free zinc ions (Zn) then activate Metal Transcription Factor 1 (MTF-1) in the nucleus to bind to transcription factor binding sites containing Metal-Responsive Elements (MRE). This induces expression of Mt and other genes. These genes may also be induced by oxidative stress, acting upon antioxidant-responsive elements (ARE) and Enhancer Box (E box). Oxidative stress may come from external oxidizing agents or from the metal itself. Finally, zinc ions may also directly activate antioxidant enzymes. Adapted from Haq et al. (2003) by Wilma IJzerman.

It must be emphasized that the model discussed above is inspired by work on vertebrates. Arthropods (Drosophila, Orchesella, Daphnia) could have a similar mechanism since they also have an MTF-1 homolog that activates Mt, however, the situation for other invertebrates such as annelids and gastropods is unclear; their Mt genes seem to lack MREs, despite being inducible by cadmium. In addition, the variability of metallothioneins in invertebrates is extremely large and not all metal-binding proteins may be orthologs of the vertebrate metallothionein. In snails, a cadmium-binding, cadmium-induced MT functions alongside a copper-binding MT while the two MTs have different tissue distributions and are also regulated quite differently.

While both phytochelatin and metallothionein will sequester essential as well as non-essential metals (e.g. Cd) and so contribute to detoxification, the widespread presence of these systems throughout the tree of life suggests that they did not evolve primarily to deal with anthropogenic metal pollution. The very strong inducibility of these systems by non-essential elements like cadmium may be considered a side-effect of a different primary function, for example regulation of the cellular redox state or binding of essential metals.

Target organs

Any tissue-specific accumulation of metals can be explained by turnover of metal-binding ligands. For example, accumulation of cadmium in mammalian kidney is due to the fact that metallothionein loaded with cadmium cannot be excreted. High concentrations of metals in the hind segments of earthworms are due to the presence of “residual bodies” which are fully packed with intracellular granules. Accumulation of cadmium in the human prostrate is due to the high concentration of zinc citrate in this organ, which serves to protect contractile proteins in sperm tails from oxidation; cadmium assumedly enters the prostrate through zinc transporters.

It is often stated that essential metals are subject to regulatory mechanisms, which would imply that their body burden, over a large range of external exposures, is constant. However, not all “essential” metals are regulated to the extent that the whole-body concentration is kept constant. Many invertebrates have body compartments associated with the gut (midgut gland, hepatopancreas, Malpighian tubules) in which metals, often in the form of mineral concretions, are inactivated and stored permanently or exchanged very slowly with the active pool. Since these compartments are outside the reach of regulatory mechanisms but usually not separated in whole-body metal analysis, the body burden as a whole is not constant. Some invertebrates even carry a “backpack” of metals accumulating over life. This holds, e.g., for zinc in barnacles, copper in isopods and zinc in earthworms.

Accumulation of metals in target organs may lead to toxicity when the critical binding or excretion capacity is exhausted and metal ions start binding aspecifically to cellular constituents. The organ in which this happens is often called the target organ. The total metal concentration at which toxicity starts to become apparent is called the critical body concentration (CBC) or critical tissue concentration. For example, the critical concentration for cadmium in kidney, above which renal damage is observed to occur, is estimated to be 50 μg/g. A list of critical organs for metals in the human body is given in Table 1.

The concept of CBC assumes that the complete metal load in an organ is in equilibrium with the active fraction causing toxicity and that there is no permanent storage pool. In the case of storage detoxification the body burden at which toxicity appears will depend on the accumulation history.

Table 1. Critical organs for chronic toxicity of metals in the human body

Metal or metalloid | Critical organ | Symptoms
Al | Brain | Alzheimer’s disease
As | Lung, liver, heart gut | Multisystem energy disturbance
Cd | Kidney, liver | Kidney damage
Cr | Skin, lung, gut | Respiratory system damage
Cu | Liver | Liver damage
Hg | Brain, liver | Mental illness
Ni | Skin, kidney | Allergic reaction, kidney damage
Pb | Bone marrow, blood, brain | Anemia, mental retardation

References

Cobbett, C., Goldsbrough, P. (2002). Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual Review of Plant Biology 53, 159-182.

Dallinger, R., Berger, B., Hunziker, P., Kägi, J.H.R. (1997). Metallothionein in snail Cd and Cu metabolism. Nature 388, 237-238.

Dallinger, R., Höckner, M. (2013). Evolutionary concepts in ecotoxicology: tracing the genetic background of differential cadmium sensitivities in invertebrate lineages. Ecotoxicology 22, 767-778.

Haq, F., Mahoney, M., Koropatnick, J. (2003) Signaling events for metallothionein induction. Mutation Research 533, 211-226.

Hopkin, S.P. (1989) Ecophysiology of Metals in Terrestrial Invertebrates. London, Elsevier Applied Science.

Nieboer, E., Richardson, D.H.S. (1980) The replacement of the nondescript term "heavy metals" by a biologically and chemically significant classification of metal ions. Environmental Pollution Series B 1, 3-26.

Rainbow, P.S. (2002) Trace metal concentrations in aquatic invertebrates: why and so what? Environmental Pollution 120, 497-507.