## 4.2.11. Toxicity mechanisms of metals

Author: Nico M. van Straalen

Reviewers: Philip S. Rainbow, Henk Schat

Learning objectives

You should be able to

- list five biochemical categories of metal toxicity mechanisms and describe an example for each case
- interpret biochemical symptoms of metal toxicity (e.g. functional categories of gene expression profiles) and explain these in terms of the mode of action of a particular metal

Keywords: Reactive oxygen species, protein binding, DNA binding, ion pumps,

Synopsis

Toxicity of metals on the biochemical level is due to a wide variety of mechanisms, which may be classified as follows, although they are not mutually exclusive: (1) generation of radical oxygen species (Fe, Cu), (2) binding to nucleophilic groups in proteins (Cd, Pb), (3) binding to DNA (Cr, Cd), (4) binding to ion channels or membrane pumps (Pb, Cd), (5) interaction with the function of essential cellular moieties such as phosphate, sulfhydryl groups, iron or calcium (As, Cd, Al, Pb). In addition, these mechanisms may act simultaneously and interact with each other. There are interesting species patterns of susceptibility to metals, e.g. mammals are hardly susceptible to zinc, while plants and crustaceans are. Earthworms, gastropods and fungi are quite sensitive to copper, but not so for terrestrial vertebrates. In this section we discuss five different categories of metal toxicity as well as some patterns of species differences in sensitivity to metals.

Generation of reactive oxygen species

Reactive oxygen species (ROS) are activated forms of oxygen that have one or more unpaired electrons in the outer orbit. The best knowns are superoxide anion (O2–), singlet oxygen (1ΔgO2), hydrogen peroxide (H2O2) and hydroxyl radical (OH•) (see the section on Oxidative stress), effective catalyzers of reactive oxygen species. This relates to their capacity to engage in redox reactions with transfer of one electron. One of the most famous reactions is the so-called Fenton reaction catalyzed by reduced iron and copper ions:

Fe2+ + H2O2 → Fe3+ + OH• + OH–

Cu+ + H2O2 → Cu2+ + OH• + OH–

Both reactions produce the highly reactive hydroxyl radical (OH•), which may trigger severe cellular damage by peroxidation of membrane lipids (see the section on Oxidative Stress). Very low concentrations of metal ions can keep this reaction running, because the reduced forms of the metal ions are restored by a second reaction with hydrogen peroxide:

Fe3+ + H2O2 → Fe2+ + O2- + 2H+

Cu2+ + H2O2 → Cu+ + O2- + 2H+

The overall reaction is a metal-catalyzed degradation of hydrogen peroxide, causing superoxide anion and hydroxyl radical as intermediates. Oxidative stress is one of the most important mechanisms of toxicity of metals. This can also be deduced from the metal-induced transcriptome. Gene expression profiling has shown that it is not uncommon that more than 10% of the genome responds to sublethal concentrations of cadmium.

Protein binding

Several metals have a great affinity towards sulfhydryl (-SH) groups in the cysteine residues of proteins. Binding to such groups may distort the secondary structure of a protein at sites where SH-groups coordinate to form S-S bridges. The SH-group is a typical example of a nucleophile, that is, a group that easily donates an electron pair to form a chemical bond. The group that accepts the electron pair is called an electrophile. Another amino acid in a protein to which metals are preferentially bound is the imidazole side-chain of histidine. This heterocyclic aromatic group with two nitrogen atoms easily engages into chemical bonds with metal ions. In fact, histidine residues are often used in metalloproteins to coordinate metals at the active site and to transport metals from the roots upwards through the xylem vessels of plants.

A classical case of metal-protein interaction with subsequent toxicity is the case of lead binding to δ-aminolevulinic acid dehydratase (δ-ALAD). This is an enzyme involved in the synthesis of hemoglobin. It catalyzes the second step in the biosynthetic pathway, the condensation of two molecules of δ-aminolevulinic acid to one molecule of porphobilinogen, which is a precursor of porphyrin, a functional unit binding iron in hemoglobin (Figure 1). The enzyme has several sulfhydryl groups that are susceptible to lead. In the erythrocyte more than 80% of lead is in fact bound to the δ-ALAD protein (much more than is bound to hemoglobin). Inhibition of δ-ALAD leads to decreased porphyrin synthesis, insufficient hemoglobin, loss of oxygen uptake capacity, and eventually anemia.

Because the inhibition of δ-ALAD by lead occurs at already very low exposure levels, it makes a very good biomarker for lead exposure. Measurement of δ-ALAD activity in blood has been conducted extensively in workers of metal-processing industries and people living in metal-polluted environments. Also in fish, birds and several invertebrates (earthworms, planarians) the δ-ALAD assay has been shown to be a useful biomarker of lead exposure. In addition to lead, mercury is known to inhibit δ-ALAD, while the inhibitions by both lead and mercury can be alleviated to some extent by zinc.

Figure 1. Formation of porphobilinogen from δ-ALA, catalyzed by δ-ALAD.

DNA binding

Chromium, especially the trivalent (Cr3+) and the hexavalent (Cr6+) ions are the most notorious metal species known to bind to DNA. Both trivalent and hexavalent chromium may cause mutations and hexavalent chromium is also a known carcinogen. Although the salts of Cr6+ are only slightly soluble, the reactivity of the Cr6+-ion is so pronounced that only very little hexavalent chromium salt is already dangerous.

The genotoxicity of trivalent chromium is due to the formation of crosslinks between proteins and DNA. Any DNA molecule is surrounded by proteins (histones, regulatory proteins, chromatine). Cr3+ binds to amino acids such as cysteine, histidine and glutamic acid on the one hand, and to the phosphate groups in DNA on the other, without any preference for a specific nucleotide (base). The result is a covalent bond between DNA and a protein that will inhibit transcription or regulatory functions of the DNA segment involved.

Another metal known to interact with DNA is nickel. Although the primary effects of nickel are to induce allergic reactions, it is also a known carcinogen. The exact molecular mechanism is not as well known as in the case of chromium. Nickel could crosslink proteins and DNA in the same way as chromium, but is also argued that nickel’s carcinogenicity is due to oxidative stress, resulting in DNA damage. Another suggested mechanism is that nickel could interfere with the DNA repair system.

Inhibition of ion pumps

Many metals may compete with essential metals during uptake or transport across membranes. A well-known case is the competition between calcium and cadmium at the Ca2+ATPase pump in the basal membrane of fish gills (Figure 2).

The gills of fish serve as a target for many water-born toxic compounds because of their large contact area with the water, consisting of several membranes, each with infoldings to increase the surface area, and also their high metabolic activity which stems from their important regulatory activities (uptake of oxygen, uptake of nutrients and osmoregulation). The single-layered epithelium has two types of cells, one active in osmoregulation (called chloride cells), and one active in transport of nutrients and oxygen (called respiratory cells). There are strong tight junctions between these cells to ensure complete impermeability of the epithelium to ions. The apical membrane of the respiratory cells has many uptake pumps and channels (Figure 2). Calcium enters the cells though a calcium channel (without energetic costs, following the gradient). The intracellular calcium concentration is regulated by a calcium-ATPase in the basal membrane, which pumps calcium out of the epithelial cells into the blood.

Figure 2. Schematic representation of the cells in a fish gill epithelium, showing the fluxes of calcium and cadmium. Calcium enters the cell through calcium channels on the apical side, and is pumped out of the cells into the circulation by a calcium ATPase in the basal membrane. Cadmium ions enter the cells also through the calcium channels, but inhibit the basal calcium ATPAse, causing hypocalcemia in the rest of the body. m = mucosa (apical side), s = serosa (basal side), BP = binding protein, mito = mitochondrion, ER = endoplasmic reticulum. Redrawn from Verbost et al. (1989) by Evelin Karsten-Meessen.

Water-borne cadmium ions, which resemble calcium ions in their atomic radius, enter the cell through the same apical calcium channels, but subsequently inhibit the basal membrane calcium transporter by direct competition with calcium for the binding site on the ATPAse. The consequence is an accumulation of calcium in the respiratory cells, and a lack of calcium in the body of the fish, which causes a variety of secondary effects; amongst others hormonal disturbance, while a severe decline of plasma calcium is a direct cause of mortality. This effect of cadmium occurs at very low concentrations (nanomolar range), and it explains the high toxicity of this metal to fish. Similar cadmium-induced hypocalcemia mechanisms are present in the gill membranes of crustaceans and most likely also in gut epithelium cells of many other species.

Interaction with essential cellular constituents

There are various cellular ligands outside proteins or DNA that may bind metals. Among these are organic acids (malate, citrate), free amino acids (histidine, cysteine), and glutathione. Metals may also interfere with the cellular functions of phosphate, iron, calcium or zinc, for example by replacing these elements from their normal binding sites in enzymes or other molecules. To illustrate a case of interaction with phosphate we discuss shortly the toxicity of arsenic. Arsenic is strictly speaking not a metal, since arsenic oxide may engage in both base-forming and acid-forming reactions. Together with antimony and four other, lesser-known elements, arsenic is indicated as a “metalloid”.

Arsenic is a potent toxicant; arsenic trioxide (As2O3) is well known for its high mammalian toxicity and its use as a rodenticide and wood preservative. There are also therapeutic applications of arsenic trioxide, against certain leukemias and arsenic is often implied in homeopathic treatments. Arsenic compounds are easily transported throughout the body, also across the placental barrier in pregnant women.

Arsenic can occur in two different valency states: arsenate (As5+) and arsenite (As3+). The terms are also used to indicate the oxy-salts, such as ferric arsenate, FeAsO4, and ferric arsenite, FeAsO3. Inside the body, arsenic may be present in oxidized as well as reduced state, depending on the conditions in the cell, and it is enzymatically converted to one or the other state by reductases and oxidases. It may also be methylated by methyltransferases. The two different forms of arsenic have quite different toxicity mechanisms. Arsenate, AsO43–, is a powerful analog of phosphate, while arsenite (AsO33–) reacts with SH-groups in proteins, like the metals discussed above. Arsenite is also a known carcinogen; the mechanism seems not to rely on DNA binding, like in the case of chromium, but on the induction of oxidative stress and interference with cellular signaling.

The most common reason of chronic arsenic poisoning is due to inhibition of the enzyme glyceraldehyde phosphate dehydrogenase (GAPDH). This is a critical enzyme of the glycolysis, converting glyceraldehyde-3-phosphate into 1,3-biphosphoglycerate. However, in the presence of arsenate, GAPDH converts glyceraldehyde-3-phosphate into 1-arseno-3-phosphoglycerate. Actually arsenate acts as a phosphate analog to “fool” the enzyme. The product 1-arseno-3-phosphoglycerate does not engage in the next glycolytic reaction, which normally produces one ATP molecule, but it falls back to arsenate and 3-phosphoglycerate, without the production of ATP, while the arsenate released can act again on the enzyme in a cyclical manner. The result is that the glycolytic pathway is uncoupled from ATP-production. Needless to say this signifies a severe and often fatal inhibition of energy metabolism.

Species patterns of metal susceptibility

Animals, plants, fungi, protists and prokaryotes all differ greatly in their susceptibility to metals. To give a few examples:

- Earthworms and snails are known to be quite sensitive to copper; the absence of earthworms in orchards and vineyards where copper-containing fungicides are used is well documented. Snails cannot be cultured in water that runs through copper-containing linings. Fungi are also sensitive to copper, which explains the use of copper in fungicides. Also many plants are sensitive to copper due to the effects on root growth. Among vertebrates, sheep are quite sensitive to copper, unlike most other mammals.
- Crustaceans as well as fish are relatively sensitive to zinc. Mammals, however, are hardly sensitive to zinc at all.
- Humans are relatively sensitive to lead because high lead exposure lead disturbs the development of children’s brain and is correlated with low IQ-scores. Most invertebrates however, are quite insensitive to lead.
- Although many invertebrates are quite sensitive to cadmium, the interspecies variation in sensitivity to this element is particularly high, even within the same phylogenetic lineage. The soil-living oribatid mite Platynothrus peltifer is one of the most sensitive invertebrates with respect to the effect of cadmium on reproduction, however Oppia nitens, also an oribatid, is extremely tolerant to cadmium.

In the end, such patterns must be explained in terms of the presence of susceptible biochemical targets, different strategies for storage and excretion, and differing mechanisms of defence and sequestration. However, at the moment there is no general framework by which to compare the variation of sensitivity across species. Also, there is no relation between accumulation and susceptibility; some species that accumulate metals to a large degree (e.g. copper in isopods) are not sensitive to the same metal, while others, which do not accumulate the metal, are quite sensitive. Accumulation seems to be partly related to a species feeding strategy (e.g. spiders absorb almost al the (fluid) food they take in and any metals in the food will accumulate in the midgut gland); accumulation is also related to specific nutrient requirements (e.g. copper in isopods, manganese in some oribatid mites). Finally, some populations of some species have evolved specific tolerances in response to their living in a metal-contaminated environment, on top of the already existing accumulation and detoxification strategies.

Conclusion

Metals do not form a homogeneous group. Their toxicity involves reactivity towards a great variety of biochemical targets. Often several mechanisms act simultaneously and interact with each other. Induction of oxidative stress is a common denominator, as is reaction to nucleophilic groups in macromolecules. The great variety of metal-induced responses makes them interesting model compounds for toxicological studies.

References

Cameron, K.S., Buchner, V., Tchounwou, P.B. (2011). Exploring the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature review. Reviews of Environmental Health 26, 81-92.

Ernst, W.H.O., Joosse-van Damme, E.N.G. (1983) Umwelbelastung durch Mineralstoffe. Fischer Verlag, Jena.

Singh, A.P., Goel, R.K., Kaur, T. (2011) Mechanisms pertaining to arsenic toxicity. Toxicology International 18, 87-93.

Verbost, P.M. (1989) Cadmium toxicity: interaction of cadmium with cellular calcium transport mechanisms Ph.D. thesis, Radboud Universiteit Nijmegen.