Introduction

Cadmium (Cd) is a nonessential heavy metal that induces adverse health effects in humans and other organisms [1]. It is now well accepted that Cd can accumulate in many organs, including the liver, kidney, heart, bone, pancreas, and testis, and it can adversely affect organ function and overall health. The most commonly used therapeutic strategy for heavy metal poisoning is chelation therapy, which promotes metal excretion. However, chelators for Cd toxicity have numerous safety and efficacy concerns [2,3,4]. The development of safe and efficient strategies to reduce Cd toxicity remains an area of ongoing research.

Dietary zinc (Zn) supplements are recognized for the important roles they play in the alleviation or prevention of Cd toxicity because Zn is readily available as a nutritional ingredient, affordable as a dietary supplement, and has not been described to have adverse side effects. Numerous studies have shown that Zn can reduce Cd toxicity. For example, Zn inhibits Cd accumulation and ROS production in Cd-treated HeLa cells and bovine aorta endothelial cells [5]. Zn supplementation improves the biochemical characteristics of the distal femur and femoral diaphysis in male rats chronically exposed to Cd [6]. Increased Zn consumption prevents alterations in lipid metabolism induced by Cd in male rats [7], and it reduces Cd-mediated hepatotoxicity in rats [8]. Zn influences several enzymes involved in DNA metabolism and inhibits apoptosis via its effect on transcription factors that are activated during apoptosis [9]. In general, the underlying mechanism of Zn protection as it relates to Cd toxicity could be divided into three activities: direct competition between these two metals, Zn-mediated MT induction, and Zn-mediated redox homeostasis. Zn and Cd have similar chemical structures and charges, and several studies have shown that Zn pretreatment reduces cellular Cd uptake; therefore, metal transporters are logical targets for further investigation [10,11,12]. Similarly, the roles of MT and redox homeostasis are important for reducing Cd toxicity. This review summarizes current knowledge related to the role of Zn and metal transporters in reducing Cd toxicity, discusses other relevant mechanisms, and presents potential future directions.

There is no agreement in the literature regarding how to treat Cd toxicity, and there are few human studies, which are typically anecdotal [13]. Available defense strategies against Cd are grouped according to their underlying mechanisms. These include antioxidant defense, mitochondrial protection, metal chelation, prevention of macromolecular damage, cytoskeletal rearrangements, hormetic response, co-exposure to other metals or trace elements, reduced uptake of Cd, removal of Cd, and altered toxicity of Cd by treatment with environmental factors [14]. Chelation therapy is commonly used clinically to treat metal toxicity [15]. Detoxification of Cd with EDTA and other chelators has been shown to be therapeutically beneficial in humans and animals [15]. However, chelation therapy is reported to have a number of safety and efficacy issues [16, 17]. Co-exposure to other metals or trace elements, such as Zn, is another possible therapeutic strategy. The mechanisms of Zn protection on Cd toxicity include direct competition between these two metals, Zn-mediated MT induction, and Zn-mediated redox homeostasis [14].

Zn mediated competition for sarcolemmal Cd uptake

Zn is an essential metal in biological systems. Zn was initially thought to cross cell membranes via iron channels; however, Zn transporters, some types of calcium channels, and DMT1 have been shown to mediate Zn uptake. Cd is a nonessential toxic heavy metal, and no Cd-specific transporters have been identified. Because Cd is chemically similar to Zn, both are positioned in Group 12 on the periodic table, both ions bind to identical macromolecular structures containing sulfur, oxygen, and/or nitrogen [18]. It is well established that Cd competes with Zn for transporter-mediated cellular entry [10,11,12, 19, 20]. For example, the Cd uptake rate is significantly lower after cellular exposure to Cd and Zn than it is after exposure to Cd alone (as demonstrated by Orchesella cincta, springtail insects) [11]. Zn supplementation counteracts acute Cd-induced nephrotoxicity in mice [21], and the Cd levels in cattle is inversely proportional to the levels of Zn. Another study revealed a protective role for Zn in counteracting Cd uptake and toxicity in the Madin-Darby bovine kidney epithelial cell line. The content of Cd in both the particulate and cytosolic fractions is decreased by Zn pretreatment in cultured vascular endothelial cells [22]. In Caco-2 cells, 200 μM Zn reduces Cd uptake by 55% [23]. Similarly, 30 µM Zn inhibits CM Cd uptake by 65% [10] and Zn reduces Cd uptake and accumulation in synoviocytes [24]. As discussed below, numerous sarcolemmal transporters and channels may be involved in Zn-mediated reduced Cd uptake, such as Zn transporters, calcium channels, and DMT1.

Zn transporters

The metal ion Zn is essential to all living cells and is present in both extracellular and intracellular fluids. Proteomic analysis suggests that nearly 10% of human proteins have the ability to bind Zn [25], and the average total cellular Zn content in mammalian cells is estimated to be between 100 and 250 μM [26]. Zn is a co-factor for many enzymes and is involved in forming and maintaining the correct three-dimensional conformation of proteins such as Zn-finger proteins. In addition to its structural and catalytic properties, labile or free Zn may function as a signaling ion exerting regulatory functions and as a second messenger to influence the activity of various enzymes and to control intracellular signaling pathways [27].

Because Zn is necessary for normal cellular function and is potentially toxic when in excess, homeostatic mechanisms have evolved to regulate the intracellular Zn concentration. The concentration of intracellular free Zn is regulated through the activity of Zn transporters and Zn-permeable ion channels and cysteine-rich metallothionein (MT) metal-binding proteins. Two families of eukaryotic Zn transporters have been identified: the ZIP (SLC39) and ZnT (SLC30) proteins [28]. Similar to Cd transporter (Table 1), Zn transporters have varied cellular distributions, transport activities, and tissue specificities (Table 2). ZIP proteins located in the sarcolemma increase Zn cytoplasmic concentration by importing Zn or by releasing Zn intracellularly vesicles. In contrast, ZnT proteins reduce the cytoplasmic Zn concentration by exporting Zn across the plasma membrane or sequestering Zn within intracellular vesicles.

Table 1 Transporters involved in Cd transport
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Table 2 Transporters involved in Zn transport
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Evidence for the role of Zn transporters in Zn uptake

Transfection of HEK293T cells with ZIP8 cDNA enhances Zn uptake by 40% [29]. ZIP8 siRNA-treated THP1 cells showed an increase in Zn content in the culture medium with a corresponding decrease within cells, suggesting that knockdown of ZIP8 reduced Zn intake [30]. ZIP14-mediated hepatic Zn accumulation was confirmed in ZIP14 KO mice and in HepG2 hepatocytes that were treated with ZIP14 siRNA [31].

Evidence for the role of Zn transporters in Cd uptake

In addition to Zn transport, both ZIP8 and ZIP14 Zn transporters have been shown to transport Cd [32]. ZIP8-mediated Cd uptake has been demonstrated by ZIP8 cRNA-injected Xenopus oocyte cultures [33] and ZIP14-mediated uptake of Cd can be competitively inhibited by Zn [34]. It is likely that other metal transporters can competitively transport both Zn and Cd, including some calcium channels and DMT1.

Evidence for the role of ZIP8 in Cd uptake

ZIP8 was originally identified in monocytes by screening cDNA transcripts that were highly induced by the Mycobacterium bovis cell wall; it was referred to as BCG-inducible gene in monocyte clone 103.10 (BIGM103) [35]. As a member of the SLC39A transporters, ZIP8 has a high affinity for divalent metal ions, including Zn2+, Fe2+, Mn2+, and the toxic heavy metal Cd. Experiments where ZIP8 capped RNAs were microinjected Xenopus oocyte cultures showed that the Km for Cd2+ is 0.48 μM, which is higher than that of Zn2+ and Fe2+ [36]. The transport characteristics of ZIP8 were further characterized using retroviral-mediated transfection of ZIP8 cDNA into mouse fetal fibroblast cultures (rvZIP8 cells), which revealed that ZIP8 is not a proton pump and is both HCO3− and energy dependent [36]. rvZIP8 cells exhibited a 10-fold increase in the rate of Cd influx and accumulation over that of control cells, and this uptake process was energy dependent and optimal at pH 7.5. To investigate Cd transport, Cd-resistant cell lines were established, including rat lung epithelial cells (LECs) [37] and Cd-resistant cells (A7 and B5), which were generated from MT-KO mouse cells [38]. These Cd-resistant cell lines displayed ZIP8 silencing and reduced cellular Cd content. ZIP8 knockdown has also been shown to be protective against Cd toxicity in a ZIP8-transfected HEK293 cell line [39] and in MT-KO mouse fibroblast cells [40]. In contrast, ZIP8 overexpression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of Cd influx and accumulation and a 30-fold increase in sensitivity to Cd-induced cell death [41]. The same result was observed in Xenopus oocytes [33]. Tumor necrosis factor-α (TNF-α) has been reported to induce ZIP8 expression in primary human lung epithelia and A549 cells, which then increases Cd uptake and cell death because of Cd-mediated apoptosis and necrosis [42]. The involvement of ZIP8 in Cd transport has been confirmed in multiple cell types; however, ZIP8 siRNA transfection does not alter Cd uptake in Caco-2 cells [43], suggesting that ZIP8-mediated Cd transport is cell type dependent.

In vivo studies revealed similar results. Cd treatment was found to cause acute renal failure and proximal tubular damage in mice overexpressing BTZIP8-3 but not in their nontransgenic littermates [44, 45]. Moreover, ZIP8 expression under normal conditions was found to be high in the liver, kidney, lung, and testis, which are all target organs of Cd toxicity.

Evidence for the role of ZIP14 in Cd uptake

Within ZIP protein family members, the amino acid sequences of ZIP8 and ZIP14 show the highest similarity, and ZIP14 shares a similar metal ion transport profile with ZIP8. However, ZIP14 has been investigated far less than ZIP8 and is presumed to play a less important role with respect to Cd-induced toxicity in the testis, kidney, and lung. ZIP14 was first identified as a Zn influx transporter in 2005 [46]. A role for ZIP14 in cellular Cd uptake was confirmed in mouse fetal fibroblasts and Madin-Darby canine kidney (MDCK) cells; further, ZIP14-mediated Cd uptake is proportional to cell toxicity, which can be competitively inhibited by Zn [34]. ZIP14 knockdown by siRNA transfection significantly reduces Cd uptake in proximal tubule cells [47]. Of interest, ZIP14 expression is downregulated in MT-KO Cd-resistant cells, which exhibit a decreased rate of Cd uptake [48]. Recent experiments have shown that transfection with a ZIP14 siRNA markedly decreases Cd uptake at lower Cd doses in Caco-2 cells [43].

Other Zn transporters involved in Cd transport

In addition to ZIP8 and ZIP14, several additional Zn transporters may be involved in Cd transport. HEK293 cells transduced with human ZIP2 reveal substrate selectivity in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+ [49]. Knockdown of ZnT3 in primary rat hippocampal neurons abolishes Zn-mediated protection from Cd-induced neurotoxicity, which indicates the involvement of ZnT3 in the mechanism of Cd-induced hippocampal neurotoxicity [50]. Another study indicated that downregulation of ZnT-1 by treatment with an siRNA resulted in regulation of the L-type Ca2+ channel, which could attenuate Cd2+ and Zn2+ permeation and alter Cd toxicity in rat hippocampal neurons [51]. While the role of Zn transporters in Cd uptake and toxicity has been well established using in vitro studies, further in vivo studies are needed to investigate the role of Zn transporters in whole organs and organisms.

The role of DMT1 in Cd toxicity

Evidence for the role of DMT1 as a Zn transporter

The divalent metal transporter 1 (DMT1), also known as NRAMP2, SLC11A2, and DCT1, is a member of the natural resistance-associated macrophage protein (Nramp) family. DMT1 was first identified in mice in 1995 [52]; it has 12 transmembrane domains, multiple membrane targeting motifs, one consensus transport motif, and two asparagine-linked glycosylation signals in an extra cytoplasmic loop. Both the N- and C-termini of the protein are located in the cytoplasm. DMT1 transports divalent cations in exchange for a single proton. DMT1 plays a crucial role in iron homeostasis and can mediate the transport of essential and toxic divalent metal ions, including Zn2+, Mn2+, Cu2+, Co2+, and Cd2+ [53]. The involvement of DMT1 in Zn transport has been shown in HEK293 cells [54] and Caco-2 cells transfected with DMT1 shRNA plasmids, which have lower Zn content compared to control cells [55]. These results confirm that DMT1 participates in active Zn transport.

Zn transported by DMT1 is likely cell type dependent, and DMT1 tends to have a lower affinity for Zn than many other metals. By using radiotracer assays and the metal-sensitive PhenGreen SK fluorophore in Xenopus oocytes, DMT1 overexpression in oocytes was shown to strongly stimulate Cd uptake, while there was only a fourfold increase in Zn uptake [56]. Recently, Shawki et al. [57] demonstrated that DMT1 is not required for the intestinal transport of Zn using a mouse model lacking intestinal DMT1. Thus, the role of DMT1 in Zn transport requires further in vitro and in vivo investigation.

Evidence for the role of DMT1 as Cd transporter

Overexpression of DMT1 in HEK293 cells was initially shown to increase time-dependent Cd uptake [58], and a follow-up study was able to increase DMT1 expression in transduced HEK293 cells by 50-fold over endogenous expression levels; the order for transport affinity in these cells was Mn2+ > Cd2+ > Fe2+ > Pb2+ ~ Co2+ ~ Ni2+ > Zn2+ [54]. Cd transport was significantly enhanced in human DMT1 RNA-injected oocytes [59] and in Caco-2 cells [60, 61], and it was suppressed in Caco-2 cells using DMT1 siRNA knockdown [62].

The role of calcium channels in Cd toxicity

Due to the similar divalent cationic properties of Ca2+, Cd2+, and Zn2+, ion channels are obvious candidates for Zn and Cd transport [63, 64]. Zn can permeate through Ca-transporting channels that belong to various families, including the voltage-gated calcium channels (VGCCs), NMDA receptors (which are primarily in neural tissues) [65], nicotinic acetylcholine receptors, and more recently, members of the transient receptor potential (TRP) superfamily [66]. The VGCC and TRP superfamilies have been more intensively studied than the others and deserve further discussion.

Evidence for the role of VGCCs as Zn transporters

In mammals, the VGCC family consists of 10 members that are divided into three subfamilies: Cav1, Cav2, and Cav3. The Cav1 subfamily members, more commonly known as L-type Ca2+ channels (LTCCs), are predominantly expressed in the heart, skeletal muscle, neurons, and endocrine cells. The Cav3 subfamily members (TTCCs) are expressed in the heart, liver, kidney, and in neurons [67]. Although VGCCs primarily transport Ca2+, they may also transport other divalent ions, including Zn [68] and Cd [69]. Zn can enter murine cortical neurons via L- and N-type voltage-gated Ca2+ channels [68], and L-type VGCC blockers (verapamil, nitrendipine, and nifedipine) inhibit Zn uptake in stimulated β-cells [70].

Evidence for the role of the TRP superfamily as Zn transporters

In mammals, the TRP superfamily includes 27 cation channels that are subdivided into six subfamilies based on sequence similarity: TRPA (ankyrin-rich protein), TRPC (canonical), TRPM (Melastin), TRPML (mucolipins), TRPP (Policystin), and TRPV (vanilloid). TRP is widely expressed in many tissues, including the intestine, kidney, placenta, and heart [71], and these channels are known to be the major pathways for Cd transport [72]. TRPV5 (also called epithelial Ca2+ channel, ECaC) and TRPV6 (also called CaT1) were reported in 1999 [73, 74] and are the two major Cd transporters in this superfamily. Several members of the TRP superfamily are Zn permeable, including TRPV5, TRPV6, TRPM3, TRPM6/7, TRPML1, and TRPA1 [66]. The overexpression of human TRPV6 in HEK293 and MCF-7 cells increases permeability for Zn2+, Cd2+, La3+, and Gd3+ [75], suggesting that TRPV6 is involved not only in Zn transport but also in the transport of other divalent heavy metal ions. HEK293 cells transfected with pTagRFP-C1-hTRPV5 or pTagRFP-C1-hTRPV6 exhibited increased Zn toxicity [72].

Evidence for the role of VGCCs as Cd transporters

The most important Ca2+ channels relevant to Cd transport are the VGCCs. While Cd entry through L-type channels in pituitary GH cells is very low relative to the levels of Ca2+ transport, this route of uptake is relevant to overall Cd toxicity, and Ca2+ channel blockers protect against Cd toxicity [76]. Increased Ca2+ in the renal tubular lumen decreases Cd accumulation in rabbit tubular cells, validating a role for Ca2+ transporters in renal Cd uptake [77]. Nimodipine, a VGCC antagonist, protects GH4C1 cells against Cd toxicity and increases the LD50 of CdCl2 from 15 to 45 μM, whereas the VGCC agonist BAY K8644 decreases the LD50 of CdCl2. These data indicate that transport of Cd through dihydropyridine-sensitive VGCCs is a major mechanism for Cd uptake and that pharmacologic VGCC blockade can provide protection against Cd toxicity [76]. In the hepatic human cell line WRL-68, the VGCC blockers nifedipine and verapamil inhibit Cd uptake by only 35%, suggesting that in these cells, VGCCs are not the predominant mechanism for cellular Cd entry [78]. In primary rat hepatocytes, diltiazem, verapamil, nifedipine, and nitrendipine inhibit Cd uptake with a 30% maximal decrease in Cd uptake [79]. In HeLa cells lacking VGCCs, Cd was unable to induce apoptosis or cellular toxicity [80], and mibefradil inhibition of T-type VGCCs reduces Cd uptake in a Cd-resistant cell line [81]. In cardiomyocytes, verapamil was shown to be the most effective Ca2+ channel antagonist, inhibiting Cd accumulation by 76%; the next most effective agents were nitrendipine (52%), diltiazem (50%), and nifedipine (29%) [10]. However, verapamil does not inhibit Cd uptake in Caco-2 cells, suggesting that there is VGCC-independent Cd uptake in these cells [23]. Nimodipine does not protect LLC-PK1 (kidney proximal tubule) or neuroblastoma cell lines from Cd toxicity [76], and it only provides partial protection in Chinese hamster ovary cells [80]. Thus, it appears that Cd uptake through Ca2+ channels varies with cell type and context, suggesting that VGCCs are involved in one pathway for Cd transport and toxicity, but there are other pathways.

Evidence for the role of the TRP superfamily as Cd transporters

The roles of TRPV6 and TRPV5 in Cd transport have been well characterized in vitro and in vivo. Mice fed a low-Ca diet showed higher intestinal mRNA levels of TRPV6 and exhibited a corresponding increase in hepatic and renal Cd accumulation, suggesting that TRPV6 may stimulate intestinal Cd absorption [82]. HEK293 cells transfected with pTagRFP-C1-hTRPV5 or pTagRFP-C1-hTRPV6 become sensitized to Cd [72], which can be inhibited by the nonspecific human TRPV6 inhibitor 2-aminoethoxydiphenyl borate (2-APB); these data support the idea that TRPV5 and TRPV6 have roles in controlling human cellular Cd uptake.

The role of metallothionein induction by Zn in reducing Cd toxicity

MTs are small cysteine-rich proteins capable of binding up to 7–12 metal ions per MT molecule; they were first described in 1957 as Cd-binding proteins in horse kidney cells [83]. MTs play an essential role in the cellular stress response, in maintaining the homeostasis of essential metals, and in the sequestration and detoxification of Cd and other heavy metals. In mammals, MTs are mainly cytoplasmic but can also be detected in lysosomes, mitochondria, and nuclei. Four MT isoforms, designated MT-1 to MT-4, have been identified, and MT-1 and MT-2 are the predominant isoforms that are expressed in most tissues [84]. MT-3 is constitutively expressed in the central nervous system, and MT-4 is primarily expressed in stratified squamous epithelium [84]. A broad range of cellular stressors, hormones, reactive oxygen species (ROS), and cytokines induce MT gene transcription [85]. Heavy metals rapidly induce MT-1 and MT-2 transcription via metal-regulatory transcription factor 1 (MTF-1) binding to metal-responsive elements (MREs) within promoter regions [86]. MTs are efficient scavengers of free radicals generated by oxidative stress [87], and MT-overexpressing and MT-KO mouse models provide validation of the role of MT in response to Cd exposure [88, 89]. Wild-type mice develop substantial Cd tolerance and a sevenfold higher LD50 than MT-KO mice [90]. Intrauterine Cd is toxic, and MT-KO pups exposed to Cd are growth restricted, confirming both a prenatal and postnatal role for MT as a major protein required for protection from Cd toxicity [91]. Repeated Cd dosing in MT-KO mice results in nephrotoxicity at one-tenth the dose required for nephrotoxicity in control mice [92], while MT overexpression protects against acute Cd lethality and hepatotoxicity [93]. In vitro engineered cardiac tissues (ECTs) generated using wild-type and MT-overexpressing neonatal cardiomyocytes display acute Cd toxicity and a partially protective role for cardiac-specific MT overexpression on Cd-induced apoptosis [88].

Cd toxicity can also be ameliorated by MT inducers, most notably Zn [92, 94, 95]. One mechanism for Zn-induced Cd tolerance is the Zn-mediated induction of MT synthesis [96]. Zn pretreatment increases MT expression and enhances resistance to Cd in MT-WT renal cells but not in Zn pretreated MT-1/MT-2 KO renal cells [97]; further, Zn protection from Cd toxicity via MT induction occurs in other tissues [95, 98]. The partial protective effect of Zn in MT-KO-derived ECTs confirms the presence of non-MT-mediated pathways in Zn-mediated protection from Cd toxicity, including direct competition for Cd and Zn for cellular uptake in bovine vascular endothelial cells [99], stellate cells [100], and murine neonatal ECTs [101]. Some animal models show a Zn-mediated protective effect related to Cd uptake and toxicity in the absence of MT induction [11] or in MT-KO mice [95]. MT induction may be directly related to Zn dosing with a dosing threshold required for MT induction [99]. Thus, the interdependence of MT and Zn on reducing Cd toxicity is likely dependent on the dose, sarcolemmal- and intracellular-specific mechanisms, and cell type.

Zn may reduce Cd toxicity by reducing ROS

ROS play an important role in Cd toxicity. The pathways involved in Cd-induced ROS are complex and multifactorial and have been well reviewed in the literature [102, 103]. The mechanisms of Cd-induced ROS can be summarized as follows. First, Cd liberates redox-active metals such as iron and copper from tightly regulated storage. Cd then displaces iron from ferritin, which in turn increases the concentrations of unbound iron ions. These free ions cause oxidative stress [104]. Second, Cd inhibits the electron transport chain, resulting in uncoupled electron flow and ROS formation [105]. Third, Cd depletes antioxidant scavengers. Glutathione (GSH) is a primary target of Cd that interacts with GSH by binding the cysteine thiolate anion. Reduced GSH activity further disturbs the cellular redox balance, which leads to enhanced production of ROS [106, 107]. Finally, Cd exposure suppresses antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [105].

Zn is a potent antioxidant [108]. Zn plays an important role in reducing levels of ROS by several mechanisms. First, Zn acts as a co-factor for important enzymes that contribute to the proper functioning of the antioxidant defense system. Second, Zn promotes the stabilization of membranes and inhibits the enzyme nicotinamide adenine dinucleotide phosphate oxidase, a pro-oxidant enzyme. Finally, Zn can induce metallothionein synthesis, which is involved in the reduction of hydroxyl radicals (OH) and in the sequestration of the ROS produced under stress conditions [109].

Overall, ROS play an important role in Cd toxicity, and Zn has a major role in attenuating the increase in ROS [108], so it is reasonable that Zn could protect cells from Cd toxicity by suppressing ROS [110,111,112]. Zn upregulates antioxidant pathways (the Nrf2 pathway) and target genes (sod1, cat, and mt2) [113], and Zn-induced protection against Cd cytotoxicity in hepatic stellate cells occurs via maintenance of normal GSH, catalase, and GSH peroxidase activities [100]. In addition, similar results have been noted in PC12 cells [114]. The requirement in MT-KO mice of normal GSH status for Zn protection against Cd-induced renal damage may provide insight into MT-independent pathways for Zn-mediated Cd tolerance [115].

Conclusion

Zn is known to reduce Cd toxicity via direct competition for Cd uptake, induction of MT, and reduction of detrimental ROS. Various sarcolemmal transport systems have been identified to play a role in Cd accumulation, including Zn transporters, DMT1, and calcium channels, all of which may represent targets that could enhance protection from Cd. Many of the published studies on Cd toxicity and Zn countermeasures come from in vitro cell lines or small animal models. A broader range of cell types and more complex in vitro tissue culture systems as well as expanded in vivo studies are required to further confirm and characterize the contribution of cell- and organ-specific transport systems to Cd transport and Cd-induced toxicity and to test current FDA-approved and novel Cd countermeasures. Phase I and Phase II clinical trials are needed to determine the safety of Zn treatment in healthy individuals, which should be followed by limited cohort studies to determine the efficacy of Zn treatment in the reduction of Cd toxicity.