Metallothionein And Cadmium Toxicology—Historical Review And Commentary

Abstract: More than one and a half centuries ago, adverse human health effects were reported after the use of a cadmium-containing silver polishing agent. Long-term cadmium exposure gives rise to kidney or bone disease, reproductive toxicity, and cancer in animals and humans. High human exposures to cadmium occur in small-scale mining, underlining the need for preventive measures. This is particularly urgent given the growing demand for minerals and metals in global climate change mitigation. This review deals with a specific part of cadmium toxicology that is important for understanding when toxic effects appear and, thus, is crucial for risk assessment. The discovery of the low-molecular-weight protein metallothionein (MT) in 1957 was an important milestone because, when this protein binds cadmium, it modifies cellular cadmium toxicity. The present authors contributed evidence in the 1970s concerning cadmium binding to MT and protein synthesis in tissues. We showed that binding of cadmium to metallothionein in tissues prevented some toxic effects, but that metallothionein can increase the transport of cadmium to the kidneys. Special studies showed the importance of the Cd/Zn ratio in MT for the expression of toxicity in the kidneys. We also developed models of cadmium toxicokinetics based on our MT-related findings. This model combined with estimates of tissue levels giving rise to toxicity, made it possible to calculate the expected risks of exposure. Other scientists developed these models further and international organizations have successfully used these amended models in recent publications. Our contributions in recent decades included studies in humans of MT-related biomarkers showing the importance of MT gene expression in lymphocytes and MT autoantibodies for risks of Cd-related adverse effects in cadmium-exposed population groups. In a study of the impact of zinc status on the risk of kidney dysfunction in a cadmium-exposed group, the risks were low when zinc status was good and high when zinc status was poor. The present review summarizes this evidence in a risk assessment context and calls for its application to improve preventive measures against adverse effects of cadmium exposures in humans and animals. 

Keywords: cadmium toxicity; metallothionein; cadmium and zinc in metallothionein; cadmium binding in blood plasma; toxicokinetic model for cadmium; kidney toxicity of cadmium; metallothionein gene expression in lymphocytes; metallothionein autoantibodies; cadmium risk assessment 

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1. Introduction

Cadmium (Cd) is a toxic metal and adverse human health effects have been known for more than one and a half centuries [1]. Governments and responsible authorities in many countries made considerable efforts to control exposures and prevent adverse health effects. However, in some countries, there is artisanal and small-scale mining (ASM) where uncontrolled exposures to cadmium and many other metals occur [2]. There is an urgent need for adequate risk assessments and preventive measures in ASM, particularly in the context of the growing demand for minerals and metals for global climate change mitigation. The present review focuses on a specific part of cadmium toxicology that is important for understanding of how toxic effects occur and how serious they will be at various exposure levels. Such information is crucial for risk assessment. In addition to the acute gastrointestinal and respiratory effects reported by clinical doctors in 1858 [1], the toxic effects of cadmium in exposed animals and humans include lung, kidney, and bone disease, reproductive toxicity, and cancer [3]. Since 1957 [4], increasing evidence has been accumulating on the role of Metallothioneins (MTs) in cadmium toxicology. MTs are low-molecular-weight cadmium-binding proteins occurring in human and animal tissues. Piscator 1964 [5] suggested that binding of Cd to MTs modifies the toxicity of cadmium. The present authors contributed evidence during the first two decades after the discovery of MTs, concerning Cd binding to MTs in tissues in relation to Cd exposure [6,7]. In addition, we described the role of MT in Cd transport and uptake in the kidney [8,9] and their likely role in modulating the interaction of Cd with intracellular targets of importance for expression of toxicity. We have continuously contributed to the knowledge about cadmium toxicology also in the last four decades and the present review and commentary summarizes our findings and gives our views on the role of metallothionein in cadmium toxicology as applied to risk assessment. Other reviews give detailed chemical properties of metallothionein [10] and detailed molecular pathways of importance for Cd kinetics and toxicity [11], not yet fully used in risk assessment.

2. Metallothioneins, Their Discovery, Isolation, and Chemical Properties 

In 1957 Margoshes and Vallee [1] published data on a Cd-binding protein in equine renal tissue, containing a high natural content of Cd and zinc (Zn). In 1960 [12] and 1961, Kägi and Vallee [13] published the first detailed characterization of the protein from horse kidneys and named it Metallothionein (MT). In 1964, Piscator [5] described that MT could be induced by Cd exposure in rabbits, and in 1972, Nordberg et al. [7] isolated three forms of MT by isoelectric focusing. The pI of these three forms were 3.9, 4.5, and 6.0, respectively. We characterized the two main forms by amino acid analysis. This and later research demonstrated that MTs are low molecular weight (about 6500 Da varying depending on the metal content), cysteine-rich metal-binding proteins. A wide variety of organisms contain these proteins, including bacteria, fungi and all eukaryotes, i.e., plant and animal species [10,14]. 

MTs are of importance for the toxicokinetics and biochemistry of essential and nonessential metals. Metallic species of Zn, Cd, mercury and copper bind to MT in clusters (see below) Other metals/metalloids such as selenium and bismuth are also bound to MT in vivo, but the exact nature of such binding has not been characterized in detail. Although they are mainly intracellular proteins, MTs have been detected in small amounts in blood and urine. MTs are determined in blood and tissues by biochemical and immunological methods [15].

Four forms of MTs, i.e., MT 1–4 have been identified. MT-1 and MT-2 are the most studied forms, expressed in most tissues and they both consist of 61 amino acids (aa). Several isoforms of MT-1 have been identified. MT-3 occurs in brain tissue, has 68 aa, and is rich in zinc. It is sometimes called the Growth Inhibitor Factor, GIF. MT-4 is expressed in keratinocytes and has 64 aa. MT-1 and MT-2 have 20 cysteine residues (30%), they contain N-acetylmethionine and C-alanine but no aromatics, no histidine. The amino acid sequence is unique and the tertiary structure displays metal clusters. MT-1 and MT-2 have two clusters A and B with four and three metals, respectively. The C-terminal is part of the A cluster and the N-terminal of the protein forms the B-cluster [16]. Zn, Cd, Hg, and Cu make up 5–10% w/w. UV absorption varies depending on the metal bound, it is (in nm) 225 for Zn-MT, 250 for Cd-MT, 300 for Hg-MT, and 275 for Cu-MT [14,17].

The link between MT and DNA for MT-1, -2, -3, -4 is related to age; fetus, newborn, and adult. Gender aspects, i.e., differences exist between men and women. MT levels are higher in the liver tissue of women than in men. In iron deficiency there is increased MT-1 in bone marrow and in kidney MT is decreased. There is genetic polymorphism, with several genes for MT located on the same chromosome. It is possible that they are coding for specific MT functions [14,17].

MT is present in the liver, kidney, urine, plasma, and blood. It serves in several functions including transport of metals e. g. Cd, Cu, Zn. Another role is in the detoxification of metals e.g., Cd, Zn, and Hg. Non-MT-bound metal species are more toxic than MT-bound metal, the latter form accumulates in tissues. MT also serves as a free radical scavenger, it serves in the storage of metals, and metabolism of essential metals and has functions related to the immune response. Metal binding to MT modifies genotoxicity and carcinogenicity [14,17]. 

The present review summarizes experimental evidence and observations in humans concerning the protein binding of Cd in blood and tissues. Because the kidney has been considered the critical organ in long-term cadmium exposures, particular attention is given to concentrations of Cd, Zn, and MT in kidneys and the appearance of tubular proteinuria. Data reviewed in the following sections are from studies performed over 50 years. All studies on animals and humans had permission from the appropriate ethical committees.

Development in MT research since 19700 s focused on purification, identification and nomenclature, characterization, molecular biology, confirmation of results in toxicology, and chemical/biochemical characterization, discussed during the first international meeting on metallothionein in 1978 [18]. The outcome of this workshop was to conclude on a terminology of the protein metallothionein.

Purification and identification of metallothionein in biological tissues caused in the beginning problems. In the 1970s, homogenization, ultrafiltration, and gel chromatography were the conventional ways of purification. It was found that storage of the 105,000 g of the supernatant at various times and temperatures influenced greatly the outcome of protein separation which is important to be aware of today. Recording of the absorbance at 250 and 280 nm, the ratio indicating the purity of MT was monitored during gel chromatography. It was found that when storage had been in room temperature, the CdMT peak appeared at a higher molecular weight than where the MT was normally eluted when samples were kept refrigerated (+5 ◦C). Gel chromatography has to be performed at such a temperature. However, by adding mercaptoethanol to the supernatant, polymerization was reversed and the MT peak was at its normal elution volume. Our preliminary attempts to study MT by polyacrylamide gel electrophoresis were unsuccessful because of the difficulties in avoiding oxidation. Storage in low-temperature freezers was useful. After a procedure of freezing the supernatants from tissue homogenates dropwise in liquid nitrogen and storage at minus 65 degrees Celsius, the distribution pattern of absorbance ratio and of the distribution pattern of cadmium was shown to be unchanged and appeared as the same for samples taken directly for protein separation. It turned out to be very effective and useful in studying Cd and MT in tissue samples with a low concentration of both. Some studies used radiolabeling of MT with Cd109, which is excellent for studying low concentrations of Cd in biological tissues [8]. Radiolabelling with Cd showed that of the seven metals one zinc always had to be part of the protein. It also explained the success in the studies of the binding of Cd to MT and the kinetics of MT in blood and in plasma. When these procedures were not used, misinterpretations and misleading data have been reported in the literature. Freeze-dried MT can be stored in hermetic vials at −80 ◦C for a very long time without oxidation of the protein. 

3. Cadmium Toxicokinetics-Role of Metallothioneins 

3.1. Cd Uptake 

Uptake of Cd from the skin into the blood is limited after dermal application. Inhalation is the main route of uptake after exposure to airborne particulate Cd in industrial environments and is also an important route for tobacco smokers. Between 7 and 40 percent of inhaled cadmium will be taken up into blood; the higher percentages are valid for soluble cadmium compounds and nanoparticulate cadmium, for example, in cigarette smoke [19]. Cd binds to MT in lung tissue and MT is induced by Cd exposure [20]. Binding to MT modifies the toxic effects in pulmonary tissues.

Studies in humans of the uptake of Cd from the gastrointestinal tract into the systemic circulation showed approximately 5 percent uptake for men and 10 percent for women. Young women with low iron stores may take up as much as 40 percent of dietary cadmium (review [3]). There are data in animals showing a similar percentage of systemic uptake of MT-bound Cd as for other chemical species of cadmium when introduced into the gastrointestinal tract, but the systemic distribution is different (see Section 3.2.) and part of ingested CdMT is taken up intact into blood. Increased uptake of non-MT cadmium from the diet occurs in animals when intake of iron, zinc, calcium or protein is low (review [3]). Experimental studies reported that a number of pathways for essential metals such as DMT 1 [21,22], CaT1 [23] and ZIP8 and ZIP14 [24] are involved in Cd uptake. Ohta and Ohba 2020 [25] cited a number of authors who had reported involvement of additional pathways in intestinal cadmium uptake (ZIP4, ZnT1, ATP7A; TRVP6) and they performed in vivo studies in animals with increasing oral Cd2+ doses and found related increased Cd concentrations and increased gene expression of MT-1, MT-2 and ZIP14, DMT1, ATP7A and TRVP6, particularly in the duodenal tissue. The exact role of these proteins/transporters for Cd uptake still is not fully clarified. 

3.2. Cd in Blood and Transport to Tissues

Adverse effects of Cd occur to a large extent after systemic distribution to various tissues such as the kidneys, the skeleton and other organs. Transfer through blood is a major route of distribution. The low concentrations in plasma in combination with insufficient sensitivity of chemical analytical methods made it difficult for a long time to perform adequate studies of the chemical concentration and the binding of Cd in blood plasma. Friberg 1952 [26] showed a long time ago that Cd is mainly found in red blood cells in experimental animals. The use of radiolabeled Cd in combination with gel chromatography offered an opportunity also to study the binding to proteins in plasma [8,27,28].

Figure 1 shows that after a single dose of ionic Cd, binding is initially predominantly to high molecular mass proteins, probably albumin, and at longer time intervals (96 and 192 h) after administration, a considerable proportion of plasma cadmium occurs at a molecular size of MT [24]. The occurrence of Cd bound to a protein of the size of metallothionein indicates an important role for this binding form for the transport of Cd to the kidney. Like other very small proteins, MT passes through the kidney glomerular membrane into primary urine. CdMT is subsequently reabsorbed into proximal tubular cells. CdMT transport from the blood to renal tubular cells is rapid and almost complete [8,29]. Other species of Cd, for example, Cd-albumin in blood plasma do not enter the kidneys to the same extent. An example is the different kidney accumulation of Cd in animals fed CdMT and other animals fed cadmium chloride [30]. Part of the CdMT enters the blood in this form, which is accumulated in the renal cortex while Cd from CdCl2 binds to albumin in blood and accumulates mainly in the liver [27]. After a single administration of Cd2+, there is a redistribution of Cd from the liver to the kidney with time (see subsequent paragraph). This redistribution is related to the time-dependent change in binding in blood plasma (Figure 1). 

As mentioned, the concentration of Cd in blood cells is considerably higher than it is in plasma. In the experiments described in Figure 1, blood cell Cd was 100 times higher than plasma concentrations at 96 h and longer. The binding of Cd in blood cells was also studied. A major part of Cd was bound to a protein with the same molecular size as MT, and not mainly to the fractions where hemoglobin was eluted [27]. Although Cd binding to the small protein in blood cells with the same size as MT, does not have an immediate impact on renal Cd accumulation, the gradual breakdown of blood cells will mean a slow release that will possibly also end up in the proximal tubules of the kidney. A role for CdMT in the transport of Cd to the kidney is presently widely accepted as a probable course of events also in humans [11], but as pointed out by Thévenod and Wolff [11], chromatographic evidence in humans is lacking. On the other hand, MT has been detected by immunologic methods in human blood sera from normal and occupationally Cd-exposed humans [31,32] and it seems likely that it would bind Cd. CdMT occurs in human urine [31] (see also Section 3.3). As mentioned in the introduction to this section, because of the limited sensitivity of methods for chemical analysis of Cd, it has not been possible to study cadmium binding to plasma proteins in humans at existing exposure levels. Recently, Li et al., 2021 [33] reported that in 11 out of 29 blood samples (average plasma Cd 0.08 ng/mL) cadmium appeared to be bound to Apo-lipoprotein A1 (ApoA1). They were unable to identify Cdbinding proteins in crude plasma samples and used procedures to remove major proteins from plasma before examining the remaining proteins. It is unclear whether these authors took precautions to avoid oxidation and polymerization of MT and it is possible that the procedures to remove major proteins influenced Cd distribution among proteins. It would be interesting to examine this possibility in future studies. 

Figure 1. Binding of cadmium in blood plasma. The panels show the results of gel-chromatographic (G75) separation (at +5 ◦C) of blood plasma from mice at various time points after s.c. injection of a single dose of radiolabeled CdCl2. (A): 20 min after injection, (B): 96 h after injection, (C): 192 h after injection. At the shorter time (20 min) all Cd appeared in a high molecular weight peak (fractions 12–14). At longer times (B,C), when the concentration of Cd in plasma was 9 nanomol/kg, a considerable proportion of plasma Cd was detected in a second peak (fractions 23–24) at the molecular size of MT. Line with dots: radiocadmium, unbroken line optical density 254 nm (OD). (Picture of Original drawing of chromatographic results. Experimental details described in [27]).

3.3. Cadmium Distribution among Organs 

After a single exposure to inorganic Cd salts in experimental animals, there is initially a high Cd concentration in the liver, decreasing with time. A redistribution occurs to the kidney and this organ later displays the highest concentration among body organs [34–36]. The increase in the kidney Cd concentration can continue for months after a single exposure. Organ distribution is dose-dependent. After high doses, regardless of exposure route, there is a larger proportion of Cd in the liver than at lower doses. At low doses, the accumulation in the kidney is more prominent, e.g., [37] Additionally, in long-term exposure, the kidney has the highest concentration of Cd [36]. Piscator 1964 [5] and Nordberg et al. [6] examined the binding of Cd in the liver tissue of Cd-exposed experimental animals and found a major proportion of Cd bound to MT. Repeated exposure to cadmium gave rise to higher levels of Cd and MT in the liver thus demonstrating that Cd exposure induced the synthesis of MT in that tissue. The authors considered that the binding of cadmium to MT is of considerable importance for cadmium toxicology. Cd exposure induces the synthesis of MT-1 and MT-2 in many tissues in animals and humans (Section 2). As mentioned (Figure 1) a proportion of blood Cd, both in hemolysate from blood cells and in blood plasma, is bound to an MT-like protein. Thus, a likely explanation for the redistribution of Cd from the liver to the kidney is a release of CdMT from the liver and transport to the kidney by glomerular filtration and reabsorption in the kidney tubules. Transport of injected CdMT, isolated from Cd-exposed animals, was quick from the blood into the kidney. Approximately 95 percent of the injected dose is taken up by renal tubules [9,29]. Uptake into proximal tubular cells occurs through the megalin: cubilin-receptor-mediated endocytosis (review [11,38]). Cd build-up in these cells stimulates MT synthesis, and a continuous rebinding to MT takes place in these cells. This explains why the biological half-life of Cd in such cells is so long. In humans, the half-life is estimated to be 10–30 years. Thus, at background exposures, Cd accumulates continuously during the human lifespan. When the concentration of Cd in the kidney cortex increases, a critical concentration is reached, and kidney dysfunction appears (see Section 4.1).

Figure 2 describes a scheme of the likely flow of albumin-bound Cd from plasma to the liver, where Cd-albumin is taken up and degraded, the released Cd2+ induces the synthesis of MT and binds to newly synthesized MT. 

Figure 2. Basic flow scheme of Cd in the body demonstrating the role of binding forms in blood and MT synthesis and degradation. aa, amino acids; Alb, albumin GSH, glutathione; MT, metallothionein. Modified from [39]

Thus, in continuous exposure, CdMT is the dominating form of Cd in the liver. Subsequently, a small proportion of liver CdMT enters plasma from where it is filtered through the glomerular membrane and taken up in kidney tubules where cellular damage may ensue (Section 4.1). First presented in 1984 by one of the present authors [39], this scheme has been widely accepted and supported by data contributed by other scientists.

Chan et al., 1993 [40] provided support for the transport of CdMT from the liver to the kidney by showing a gradual uptake of Cd in the kidneys after transplantation of Cd-containing livers to non-Cd exposed rats. Liu et al., 1996 [41] and Liu and Klaassen 1996 [42] showed differences in Cd kinetics between transgenic (MTnull) and wild-type mice. In MTnull mice, the elimination of Cd was much faster than in wild-type mice. Cd concentrations in the kidney increased with time in wild-type mice, but not in MTnull mice. These observations support a role of MT in tissue retention and transport of cadmium to the kidney. A review of the presently available evidence, giving general support to the explanatory scheme described in Figure 2, but including detailed and up-to-date information on biochemical pathways explaining Cd kinetics and toxicodynamics was presented by Sabiolic et al. [43]-the reader is referred to this review for further details.

As a result of MT binding and the mentioned time-related distribution changes, adult humans with long-term, low-level exposures, e.g., background exposures in Sweden, have 50 percent of their Cd body burden in the kidneys. In the kidney, the highest Cd concentration is in the kidney cortex (review [3]) 

3.4. Cadmium Excretion-Biological Half-Life 

Cd induces MT synthesis in the liver, kidney, and other tissues (Sections 1 and 2) and a large proportion of tissue Cd is bound to MT and becomes trapped in the tissues in this form. This explains the long biological half-lives of cadmium in the tissues of humans and animals. Only 0.01–0.02 percent per day of the Cd body burden is excreted in urine and feces. The biological half-life of Cd in human tissues is very long in the phase of Cd accumulation in the kidney. When the Cd level in the kidney cortex reaches a concentration that causes renal tubular dysfunction (see Section 4.1), urinary Cd excretion increases dramatically and the half-life of Cd in the kidney decreases. 

In the accumulation phase, the half-life in human tissues such as muscles, kidney cortex and the liver is 10–30 years according to estimates based on studies of human tissues and excretion patterns. In blood, there is a fast component (100 days) and a slow component (7–16 years) describing the decreasing levels after cessation of occupational exposure in humans. In animals, the half-life in blood plasma changes from minutes immediately after exposure to days at later observation times (reviewed in [3]). Akerstrom et al., 2013 [44] reported a half-life in the human kidney cortex of 23 years at a kidney cortex Cd concentration of 8 mg/kg and 43 years at 23 mg/kg. The longer retention probably is related to more efficient induction of the synthesis of MT at the somewhat higher Cd levels. Cd concentrations in the kidneys of older humans decrease after age 60, possibly because of the less efficient MT induction in older age groups. 

Urinary Cd excretion takes place by transfer of Cd from the renal tubules into urine and by excretion of a small proportion of glomerular filtrate that is not taken up into renal tubular cells as indicated by animal experiments (reviewed in [3] and Section 3.2). In the accumulation phase, before renal tubular damage is induced, urinary Cd is a good indicator of kidney and body burden of Cd. When a toxic cadmium level is reached in the kidney tubules, tubular reabsorption will be impaired and urinary excretion of Cd will increase dramatically. The relationship between kidney Cd and urine Cd changes when tubular dysfunction is induced. Urinary Cd is to a considerable extent MT-bound both in the accumulation phase and when there is tubular dysfunction [31,45–47] see also Section 4.2)

3.5. Toxicokinetic Model of Cadmium Accumulation in Kidneys

Although a discussion is ongoing on whether adverse effects of Cd on the kidneys or the skeleton should be considered as critical effects, i.e., effects that occur at the lowest external exposures, effects on the kidneys occur at low exposures and are still considered critical effects [19]. Kjellstrom and Nordberg 1978 [48] presented a toxicokinetic, multicompartment model for Cd kinetics and accumulation in the kidneys based on the identification of a crucial role of MT, largely as described in Figure 2 (see also [49]). Choudhury et al., 2001 [50] developed this model further, using later evidence. The amended model in combination with calculations based on the distribution of the critical concentration of Cd in the kidney cortex has been successfully used in risk assessments by Diamond et al., 2003 [51], ATSDR 2012 [52] and the International Union on Pure and Applied Chemistry 2018 [19]. In the latter document, these model calculations provided a perspective on findings in epidemiological studies. The calculations show that the lowest exposure level of Cd giving rise to kidney dysfunction is very low. 

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