The Role of Metal-Binding Proteins, Metallothioneins (MTs)
Metallothioneins (MTs) are heat-stable, metal-binding proteins of low molecular weight (ca. 7 kilodaltons), high cysteine content (ca. 30%), and no aromatic amino acids. They occur throughout the animal kingdom as well as in plants, and are located mainly in the cytosol or in the nucleus, the amounts of which were dependent upon the tissue metal concentration. Two or more isomers of MTs may even exist in the same animal. Affinity for MTs is metal-dependent and correlated with the distribution of metal binding sites on the MTs as well as the stoichiometry of the different types of MTs. These differences in binding strength are relevant for the involvement of MTs in metal-metal interactions.
MTs can be induced by exposure to elevated levels of Hg, Cu, Zn and Cd, and have a high affinity for these metals, although transition metals have differing affinities for MTs (in decreasing order, Hg>Cu>Cd>Zn) and displacement reactions may occur dependent on the relative concentrations of these metals (Engel and Brouwer, 1987). All MTs may be induced as a result of cadmium exposure and are capable of binding cadmium. Detoxification often involvs binding to proteins such as MTs or forming insoluble metaliferous granules. Some MTs may be basally expressed and play a role in copper and zinc metabolism and regulation (Engel and Brouwer, 1987).
MTs play a dual role in most aquatic organisms. For essential metals, MTs may be important in controlling metabolically available concentrations by binding the metals in a nontoxic unavailable form until they are required for various metabolic processes, although regulatory and/or detoxification functions may overlap. Furthermore, their function has probably pre-adapted them to act as regulators in a detoxifying role for excess concentrations of copper and zinc (Engel and Brouwer, 1987). For non-essential metals, there is no evidence that
MTs have any cadmium regulatory capacity as determined by tissue or body concentration. Cadmium detoxification relies heavily on the ability of induction and binding to MT and subsequent available sequestration capacity, which was followed within the context of cadmium uptake dynamics and distribution within organisms and correlated with increased tolerance to this metal. MT induction can not be considered as an overriding principal of cadmium tolerance, which has several other modifying factors. For example, cadmium sensitivity may have as much to do with metal competition and the capacity of MT production, if the spillover theory is invoked as a reasonable basis for delineating tolerance (Wright and Welbourn, 1994).
Critical Body Residue (CBR)
Similar to the external concentration, only a portion of total metal body burden is biologically available for interaction with sites of toxic action. Toxicity is related to the metabolically reactive pool rather than to the total internal metal burden, the capacity of which has an upper limit (Figure 10). As the basis of internal metal sequestration over different organs and tissues, it is necessary to investigate subcellular metal partitioning for better understanding mechanisms of accumulation and toxicity using a fractionation procedure, which can isolate metal-rich granules and tissue fragments from intracellular and cytosolic fractions.
Metal-rich granules (MRG) and tissue fragments can be separated from intracellular (nuclear, mitochondrial, and microsomal) and cytosolic fractions (i.e., MTs and heat-sensitive proteins) through the fractionation method of different centrifugation steps (Wallace et al., 2003). The fractionation constituents reflect the metal-binding preferences by different ligands, and the underlying principle is that metals entering the body in a reactive form are first captured by reversible (labile) binding to proteins and other ligands, followed by localization to targets that have a stronger affinity for metals.
The amount of metal compartmentalized in cytosolic (such as organelles and microsomes), heat denaturated protein (enzymes), and tissue fractions (such as nuclei, cell membranes, tissue and intact cells) seems to be most indicative of toxic pressure, which is supposed to be indicative of the excess metal fraction shown in Figure 10. The fractions such as granules, lysosomes and heat stable proteins (MTs) are maybe more important for uptake, elimination or tropic transfer than for toxic action.
The Critical Body Residue (CBR) is defined as the threshold concentration of a substance in an organism that marks the transition between no effect and adverse effect, based on assumption that the total body concentration is proportional to the concentration at the target or receptor, the effect is proportional to the concentration of metal bound to the target site, and that this target site (biotic ligand) is in direct contact with the external (aquatic) environment, which integrates internal transport and metabolism processes and toxicity at specific sites of toxic action, and provides a better understanding of the internal compartmentalization of metals in organisms and its consequences for toxicity (Vijver et al., 2005).
Cadmium ions in excess of metabolic requirements and storage capacity are potentially toxic and must be removed from the vicinity of important biological molecules, although organisms can minimize accumulation of reactive metal species at the cellular level. Therefore, the capacity of internal sequestration has a huge impact on the sensitivity of an organism to cadmium. At least two major types of cellular sequestration can occur after increased exposure to cadmium and affect their toxicokinetic availability to organisms (Table 2).
The first mechanism of cellular sequestration involves forming distinct inclusion bodies of metal accumulation, such as granule types originating from the lysosomal system and containing mainly acid phosphatase and accumulating cadmium identified by x-ray microanalysis. The second type preventing cadmium toxicity is a cytoplasmic process involving a specific metal-binding protein (heat-stable proteins, MT), because binding to MT generally can reduce the availability of toxic metals.
Table 2. Various cellular cadmium species in an organism after partitioning the total internal burden
|
Cadmium species |
Example |
|
Free ionic form or complexed ion species |
 |
|
Bound to low molecular weight and functional proteins |
hemoglobin, hemocyanine |
|
finger proteins |
|
|
Bound in the active center of enzymes |
cytochromes,carbonic anhydrase, |
|
superoxide dismutase |
|
|
Bound to low molecular weight organic acids |
citrate |
|
Bound to metallothionein, to transport proteins or other sequestration proteins |
ferritin |
|
Bound in vesicles of the lysosomal system |
intracellular granules |
|
Precipitated in extracellular granules, mineral deposits, residual bodies, and exoskeletons |
|
|
Bound to cellular constituents potentially causing dysfunction |
enzymes, ion channels, DNA |
Links to Toxicity
Relationships between bioaccumulation and adverse effects are complex when different species are compared in different environments for different metals. Although sublethal toxic effects such as reproductive impairment could be observed when bio-accumulated metals were coincident with an increased concentration. Defining adverse ecological effects, several aspects should be considered that link bioaccumulation (internal metal exposure), internal metal reactions, and the hierarchy of biological responses. Accumulation of bioactive metal is more important than total bioaccumulation, and understanding toxicity requires consideration of more than just total metal accumulated in tissues.
Taking the biotic ligand model (BLM) theory as an example, which mainly linked metal speciation in solution with the amount of metal bound at the gill surface of fish, based on the fact that such binding involves competition between metals binding to the gill binding sites (biotic ligand) and other complexing agents in solution as well as competition for the sites by hydrogen ions, calcium and magnesium. The proportion of binding sites on the gill that are occupied by metal determines the degree of disturbance to ion regulation (Figure 6). Adverse effects are determined not only by the amount of metal that is bio-accumulated, but the toxic "action" at the target site(s). For organisms other than fish, the theoretical basis is that total body residues directly determine the effect, which in organisms is assumed to be directly related to the amount of metal bound to the external body surface. The toxic effects of tested organisms can be predicted at the moment, but is restricted to some small organisms that quickly achieve an internal equilibrium for the metals between body surface and environments (Rainbow, 2002).
The sub-cellular partitioning model (SPM) taking into account the cellular fates of metals was proposed to predict metal toxicity in aquatic organisms. It considers how metal accumulation and subsequent redistribution are directly related to metal toxicity and takes advantage of a recently introduced conceptual model that groups individual subcellular fractions (e.g., metal-rich granules [MRG], cellular debris, organelles, heat-denatured proteins [HDP], and heat stable protein [HSP]) into ecotoxicological relevant compartments. For instance, organelles and HDP are grouped as the metal-sensitive fraction (MSF), and HSP and MRG are grouped as the biologically detoxified metal (BDM). The correlations between metal toxicity and subcellular metal distribution of several aquatic organisms (e.g., phytoplankton, bivalves, and fish) have been tested (Miao and Wang, 2006, Wang and Wang, 2008).
The subcellular fractionation approach may be more useful in explaining the sublethal (chronic) cadmium toxicity than the acute toxicity. When the toxicity results are applied to field situations, it is expected that they should be sufficiently conservative to protect most species (e.g., 95%).
Species-Specific Effects
The physicochemical properties of cadmium and the physiology of the organism both influence cadmium uptake, distribution, tissue accumulation, and excretion, although a diversity of specific cadmium accumulation strategies is known at the level of organs and tissues in an organism, tissue- and organ-specific. Toxicity is ultimately determined by cellular mechanisms of cadmium accumulation (Vijver et al., 2005).
Cadmium accumulation patterns are variable among species, which include regulation of body concentrations of cadmium by some species, and vastly different concentrations among species and environments. The compartmentalization or sequestration of cadmium by invertebrates is also dependent upon many factors, such as organism life history, and cadmium pre-exposure, so the modes of toxic action that sequester the bio-accumulated internal cadmium concentration are species-specific. In summary, bioaccumulation varies widely among taxa, often reflecting basic differences in biology.
Crustaceans
In crustaceans, soluble cadmium is probably largely accumulated through the gills, where it may reach high concentrations and result in tissue damage. No evidence showed that crustaceans were able to regulate their body cadmium concentration relative to ambient levels, although copper and zinc were substantially regulated. Although some crustacean MTs may have a specific copper regulatory function, all have very reactive cadmium binding sites and even, more labile of cadmium loci may be present in some additional cases. In some instances this binding capacity may result in the accumulation of very high cadmium concentrations in the hepatopancreas of crustaceans, which generally contains between two and four MT isomers.
Much of the cadmium accumulated by aquatic invertebrates is bound to MTs in the cytosol of the organ predominantly used for accumulated cadmium storage. In certain circumstances of severe cadmium exposure, there is indirect evidence that cadmium from MTs may be deposited in an insoluble form in lysosomal residual bodies. If the cells containing these lysosomal residual bodies line a tract with external access, then there is the potential for any cadmium-rich cell inclusions to be excreted, offering potential for the final accumulation pattern to be identified. The exoskeleton also has been shown to be a significant site of cadmium deposition in crustaceans, although the degree to which this occurs via the hemolymph or through external adsorption remains open to question (Wright and Welbourn, 1994).
In the shore Green crab Carcinus maenas, cadmium becomes associated with haemolymph proteins, but is rapidly turned over to the hepatopancreas where it may be stored as inorganic granules or associated with MT in proportions that depend on the degree of cadmium exposure.
Molluscs
In the bivalve molluscs, cadmium is principally accumulated via the gill from water having low environmental cadmium concentrations and can be rapidly bound into a non-toxic complex of mollusc tissues such as MTs, but retained within the body and excreted very slowly. Harmful effects may follow when the cadmium binding sites are saturated, because accumulation occurs in tissues which may be sites of toxic action (Wright and Welbourn, 1994).
Within the gill, the role of granular amoebocytes in cadmium binding has been clarified. Some researchers suggested that the cadmium-binding proteins in amoebocytes of cadmium-exposed oysters (Crassostrea gigas) were present and MTs in the cytosol of Crassostrea virginica gills that progressively sequestered cadmium at the expense of the granular fraction was located.
As for the marine mussel Mytilus edulis, kidney seems to be a major target organ for cadmium, where it may be sequestered in lysosomes resulting in granular concretions. When the kidney tissue of Bay scallop (Argopecten irradians) was exposed for 5 days to 700 ^g Cd/L, the concretions or granules contained 60% of the accumulated cadmium. However when Mytilus edulis was exposed to 100 ^g Cd/L for 3 months, even 85% of the cadmium in membrane-limited granular structures may have been associated with MTs, sulfur and sometimes phosphorus in membrane-bound vesicles. The cadmium excretion rate in Mytilus edulis was 18 times slower than the uptake rate and 50% of accumulated cadmium in Crassostrea virginica was lost in 60 days in clean water. Mussels loaded with 564 mg Cd/kg dry weight lost 47 mg/kg in a 42-day depuration period, in which time the fraction bound to MT rose from 22 to 78%.
Other researchers, also, found that the cadmium half-life in Saccostrea echinata was very long.
The granular fraction of scallops declined following three week exposure as the metal became preferentially bound to intracellular thiol groups (MTs), and high concentrations of cadmium have been found in the digestive gland bound to both high molecular weight proteins and MTs.
Fish
In fish, tissue cadmium accumulation is largely associated with MTs binding, and in contrast to invertebrates, granular concretions seem to play an insignificant role in cadmium sequestration. Cadmium has been shown to affect several enzyme systems, including those involved with neurotransmission, trans-epithelial transport, intermediary metabolism (sometimes in vitro) exposures to high cadmium concentrations.
For plaice (Pleuronectes platessa) and dabs (Limanda limanda), cadmium accumulates in the liver and gills rather than in their muscle and liver concentrations began to increase only after 70 days exposure to 5 ^g Cd/L. l09Cd pulse experiments indicate a biphasic transfer of cadmium from gills to kidney and liver, where there is little subsequent release. In minnows (Phoxinus phoxinus) and other species, cadmium was rapidly accumulated in the head region from water by the gills, probably resulted from localization in the olfactory mucosa, and the gut mucosa did not appear to be a major site of cadmium uptake from water, although this may change according to diet. However in several species of fish, the gut contained "intestinal corpuscles" – a mixture of mucous cells, mucous, and granules – which had a high cadmium concentration.
In the stone loach Noemacheilus barbatulus, cadmium sensitivity decreased as a result of higher availability of binding sites on existing MTs, compared with the rainbow trout Salmo gairdneri, where cadmium binding to existing and induced MTs was limited by relative cadmium, zinc, and copper concentrations in several tissues, particularly the liver. When plaice was injected with a cadmium dose of 100 ^g/kg, MT induction exceeded the sequestration capacity of the induced MT and led to reduced production of the enzyme ethoxyresorufin o-deethylase (EROD) and of MT itself (Wright and Welbourn, 1994).
Polychaeta
In the polychaete Neanthes arenaceodentata, MTs had no capacity to perform any homeostatic function with respect to cadmium, the binding of which seems to be a passive function of the number and affinity of available binding sites, although this species apparently possesses some capacity to adjust cadmium uptake and efflux to reduce long-term accumulation.
