New Insights into the Roles of ZIP8, a Cadmium and Manganese Transporter, and Its Relation to Human Diseases

Abstract

ZIP8, a Zrt-/Irt-related protein encoded by Slc39A8, was originally discovered as a zinc transporter, but since then its roles as a transporter for cadmium (Cd) and manganese (Mn) have also been well characterized. ZIP8 is highly expressed in the S3 segment of the proximal tubules of the mouse kidney and may play a significant role in reabsorption of both toxic Cd and essential Mn from the lumen to the epithelial cells of the proximal tubule. In recent years, associations between various human diseases and genetic variations of ZIP8 have been reported. Missense mutations in the human SLC39A8 gene are associated with serious disorders of Mn metabolism, showing symptoms similar to congenital glycosylation deficiency. Enhanced excretion of Mn via bile or urine might be the cause of extremely low blood Mn levels in ZIP8-mutated patients, leading to the defects in Mn-dependent glycosylation. Several genome-wide association studies have demonstrated the associations of multiple diseases and ZIP8 SNPs constituting missense mutations. These findings suggest that ZIP8 plays more important roles than previously expected as a modulator of Mn homeostasis in the body. Elucidation of biochemical mechanisms regarding the metal-transporting ability of ZIP8 and its alteration by mutation is required for better understanding of the role of ZIP8 in human diseases.

1. INTRODUCTION

During the last few decades, the roles of metal transporters in tissue deposition of toxic and essential metals have begun to be elucidated. Progress in this field has been achieved mainly by identifications of specific transporters for each metal. For example, understanding of the mechanisms of iron metabolism disorders such as haemochromatosis dramatically accelerated after the discovery of the iron transporter, divalent metal transporter (DMT1).¹⁾ The transport mechanisms of toxic metals such as cadmium (Cd) have been unknown for a long time, but the identification of ZIP8, a Zrt-/Irt-related protein encoded by Slc39A8, has promoted the understanding of Cd transport at the cellular level.^2–5) ZIP8 also has an affinity for divalent manganese (Mn²⁺).⁶⁾ Recent findings regarding the association of human diseases with Mn transport disorders due to mutations in human SLC39A8 gene have highlighted the important roles of ZIP8 in the maintenance of Mn homeostasis in the human body.^7,8)

In this review, we first described the functions of ZIP8 as a transporter for cellular incorporation of Cd and Mn. Secondly, the roles of ZIP8 in the kidney proximal tubules as a modulator of reabsorption of Cd and Mn were discussed. Finally, we described the human cases of Mn metabolism disorder caused by ZIP8 mutation and discussed the roles of ZIP8 in the maintenance of Mn homeostasis in the body.

2. ZIP8 AS A CADMIUM TRANSPORTER

Cd accumulates in the kidney and causes proximal tubular dysfunctions. Numerous studies have been conducted on the biological effects and in vivo dynamics of Cd, but the mechanisms by which Cd is incorporated into and excreted by cells remained obscure for a long time. Metallothionein (MT) is known as a factor affecting tissue deposition and the toxicity of metals including Cd.⁹⁾ MT is efficiently induced by Cd, binds to Cd with a high affinity, and thereby reduces the toxicity of Cd.¹⁰⁾ Thus, MT is an important modulator of the intracellular concentration, chemical form and cytotoxicity of Cd, but because of this distinct feature, it is difficult to evaluate precisely the uptake and excretion rates of Cd in the presence of MT within cells. Typically, drug transporters are identified by the development of cells that are resistant to the drug, but most of the Cd-resistant cells established so far have shown enhanced expression of MT, and little information regarding Cd transport has been provided by examination of Cd-resistant cells.^11,12)

To overcome this problem, we developed Cd-resistant Cdr-A7 and Cdr-B5 cells by a stepwise increase in Cd concentration in the media of immortalized mouse embryonic fibroblast (MEF) cells derived from MT-I and MT-II knockout mice.¹³⁾ In the absence of MT, the established Cd-resistant cells showed a reduced accumulation of Cd by about 90% compared with the parental cells. We found that the uptake rate of Cd²⁺ was remarkably suppressed in MT-null Cd-resistant cells. Since the uptake rate of Mn²⁺ was also reduced, it can be assumed that the expression of a transporter having an affinity for Mn²⁺ is responsible for the uptake of Cd²⁺ and is suppressed in MT-null Cd-resistant cells.^4,14) The comparison of gene expression between Cd-resistant and parental MT-null cells by DNA microarray analyses and subsequent real-time RT-PCR analyses revealed that the zinc (Zn) transporters ZIP8 and ZIP14, encoded by Slc39a8 and Slc39a14 respectively, were markedly down-regulated in Cd-resistant cells.^3,4) The introduction of ZIP8 small interfering RNA (siRNA) to the parental cells resulted in a remarkable decrease in the uptake of Cd²⁺.⁴⁾ These data suggest that ZIP8, which may be a transporter for Mn²⁺, is involved in the cellular uptake of Cd²⁺.

The role of ZIP8 as a Cd transporter has also been proved by another study regarding the strain difference in Cd-induced testicular hemorrhage in mice. Dalton et al. found that ZIP8 expression in the testes was evident in Cd-sensitive strains (D2, 129/svJ) and defective in Cd-resistant strains (B6, A/J) of mice.¹⁵⁾ ZIP8 overexpression was shown to increase Cd accumulation in Xenopus oocytes as well as in mice. ZIP14, which has the highest homology to ZIP8 among ZIP family members, was also shown to have an affinity for Cd²⁺.^16,17) It is now recognized that ZIP8 and ZIP14 are transporters for cellular uptake of Cd²⁺.^16,17)

It seems likely that ZIP8 has a more important role in the toxicity manifestation of Cd than ZIP14. We found that the LC₅₀ value of CdCl₂ in rat basophilic leukemia-derived RBL-2H3 cells was as low as that of MT-null cells.⁵⁾ The high sensitivity of RBL-2H3 cells to Cd was due primarily to the high accumulation of Cd compared with other rat cell lines. The expression of ZIP8 in RBL-2H3 cells was the highest among the rat cell lines examined, and the introduction of the siRNA of ZIP8, but not that of ZIP14, to RBL-2H3 cells resulted in reduced accumulation of Cd.⁵⁾ We established Cd-resistant RBL-2H3 (RBL-Cdr) cells, in which Cd accumulation was significantly reduced due to the down-regulation of ZIP8. No change in ZIP14 expression was observed in RBL-Cdr cells. Moreover, we also established Cd-resistant P⁺ cells from MT-expressing MEF cells, and found that the expression of ZIP8, but not ZIP14, was suppressed in P⁺ cells.^5,18) Thus, ZIP8 has been suggested to play a more crucial role in the transport and toxicity manifestation of Cd than ZIP14, although both Zn transporters have an affinity for Cd²⁺.

3. ZIP8 AS A MANGANESE TRANSPORTER

Before the identification of ZIP8 and ZIP14 as Cd transporters, we realized that a cellular transport system for Mn²⁺ uptake is involved in Cd²⁺ uptake since the application of the multi-tracer technique revealed that the uptake rates of Mn²⁺ were markedly decreased in Cd-resistant Cdr-A7 and Cdr-B5 cells.¹⁴⁾ Competitive inhibition of cellular uptake of Cd²⁺ and Mn²⁺ has been reported in many studies using various types of cell lines.^14,17,19) After the identification of ZIP8 and ZIP14 as Cd transporters, their abilities for the cellular uptake of Mn²⁺ were proved by experiments using mouse fibroblast cells and Xenopus oocytes in which ZIP8 or ZIP14 was overexpressed.^2,20)

RBL-2H3 cells, which show a high sensitivity to CdCl₂ due to high expression of ZIP8, also show a high sensitivity to MnCl₂ due to the high accumulation of Mn.⁵⁾ Suppression of ZIP8 expression by siRNA transfection in RBL-2H3 cells resulted in a decrease of the uptake rate of Mn²⁺ as well as Cd²⁺. We established Mn-resistant RBL-2H3 (RBL-Mnr) cells by a stepwise increase in MnCl₂ concentrations in the media and found that ZIP8, but not ZIP14, was down-regulated in RBL-Mnr cells, resulting in reduced accumulation of Mn.¹⁸⁾ RBL-Mnr cells showed a cross-resistance to CdCl₂; likewise, RBL-Cdr cells, the ZIP8 down-regulated cells, showed a cross-resistance to MnCl₂, both resistances being acquired primarily by the reduced accumulation of Mn.¹⁸⁾ Cd-resistant P⁺ cells, in which ZIP8 but not ZIP14 was down-regulated, also showed a cross-resistance to MnCl₂.⁵⁾ All these results suggest the important role of ZIP8 but not ZIP14 in the cellular uptake of Mn²⁺, although both Zn transporters have an affinity for Mn²⁺.

4. ROLES OF ZIP8 IN KIDNEY

Because Cd causes injuries in the proximal tubules of the kidney, we investigated the expression and roles of ZIP8 in the kidney. In situ hybridization of the mouse kidney showed that mRNA of ZIP8 is highly detected in the boundary region between the cortex and outer medulla in the proximal tubules, where the S3 segment (straight parts before the Henle loops) of the proximal tubules are concentrated. Briefly, the proximal tubule from the glomerulus to Henle’s loop is divided into three segments: S1, S2, and S3.²¹⁾ It is known that endocytic reabsorption of low-molecular-weight proteins including MT is carried out mainly in the S1 and S2 segments, while the S3 segment is rich in transporters including those involved in the absorption of glutathione. In addition, proximal tubule epithelial cells (PTECs) are characterized by their polarity. The expressions and functions of transporting systems are definitely different between the apical side, which faces the lumen, and the basal side, facing the blood vessels.

To test the roles of ZIP8 in renal cells, we utilized a trans-well cell culture system, in which the uptake and excretion of substances at the apical and basolateral sides of cells can be measured separately.²¹⁾ When PT cells, the immortalized mouse PTECs, were cultured in the trans-well system, the cells developed tight junctions with sufficiently high values of transepithelial electrical resistance. We found that the apical uptake of Cd²⁺ was competitively inhibited by Mn²⁺ and Zn²⁺, and the uptake of Mn²⁺ was inhibited by Cd²⁺ and Zn²⁺. The introduction of ZIP8 siRNA into PT cells resulted in roughly 50% decreases in the uptake rates of Cd²⁺ and Mn²⁺ from the apical sides of the cells. These results suggest that ZIP8 plays an important role in the uptake of Cd²⁺ and Mn²⁺ from the proximal tubule lumen into PTECs. Since our in vivo study showed that ZIP8 is highly expressed in the S3 segment, we proposed a model of renal handling of Cd; Cd-MT is mainly reabsorbed in the S1 and S2 segments via endocytosis, but the portion of Cd²⁺ that is released into the lumen of the proximal tubule from the S1 and S2 segments or filtered through the glomerulus could be reabsorbed in the PTECs in the S3 segment, probably via ZIP8.²¹⁾ To confirm this hypothesis, a study with immortalized PTECs derived from each segment of S1, S2, and S3 is now in progress at our laboratory.

The suppression of ZIP8 also reduced the apical uptake of Mn²⁺ in PT cells cultured in the trans-well system, suggesting an important role of ZIP8 in the uptake of luminal Mn into kidney PTECs.²¹⁾ Since Mn cannot be bound to MT, the uptake of Mn by metal transporters such as ZIP8 could be the sole route of renal Mn reabsorption. Thus, ZIP8 in the kidney may play an important role in the maintenance of the homeostasis of Mn, an essential element, in the body.

5. HUMAN DISEASES WITH ZIP8 MUTATIONS

Recently, human cases with SLC39A8 gene mutations have been identified. In Germany, an infant showing symptoms of cranial asymmetry, convulsions, and dwarfism, which are similar to those observed in congenital glycosylation deficiency, was found to have mutations in the SLC39A8 gene.⁷⁾ Missense mutations were found at two sites: glycine at 38 to arginine and isoleucine at 340 to asparagine (Table 1). Another patient was found to have mutations in SLC39A8 at different sites: valine at 33 to methionine and serine at 335 to threonine.⁷⁾ In Egypt, patients with similar symptoms were found to have a mutated SLC39A8 gene, causing yet another variant of ZIP8, with a change in the amino acid at a single site (glycine at 38 to arginine).⁸⁾ In these patients, blood Mn concentrations were undetectable or very low. Since no mutations in other enzymes for glycosylation were detected in these patients, it is presumed that Mn deficiency due to the ZIP8 mutation resulted in a defect in the enzymatic activity of galactosyltransferase, which is known to be completely dependent on Mn for its activity.

Table 1. Symptoms and Mutations in SLC39A8

Patient information	Symptoms	Mutations	References
German child	Low Mn level in blood, cranial asymmetry, convulsion, dwarfism	c. 112G > C (p. Gly38Arg)	Park et al., Am. J. Hum. Genet. (2015)
		c. 1019T > A (p. Ile340Asn)
German child	Psychomotor disorder, cerebellar atrophy, strabismus, scoliosis	c. 97G > A (p. Val33Met)	Park et al., Am. J. Hum. Genet. (2015)
		c. 1004G > A (p. Ser335Thr)
Egyptian children	Cerebellar atrophy, intellectual disorder, developmental delay, hypotension, strabismus	c. 112G > C (p. Gly38Arg)	Boycott et al., Am. J. Hum. Genet. (2015)
Brothers in Lebanon	Leigh syndrome, developmental disorder, dystonia, Low Mn level in blood/urine	c. 339G > C (p. Cys113Ser)	Riley et al., J. Inherit. Metab. Dis. (2017)

Another case of ZIP8 mutation at a different site (Cys113Ser) was found in a patient in Lebanon with congenital glycosylation disorder type II (CDG) showing mental retardation and cerebellar atrophy, a characteristic of a mitochondrial disease called Leigh’s syndrome²²⁾ (Table 1). Although the actual relationship between ZIP8 mutation and this syndrome remains unknown, it is presumed that a defect in Mn-dependent superoxide dismutase (Mn-SOD) in the mitochondria might be involved.

These manifestations of disease in association with serious disorders in Mn metabolism in patients having mutant ZIP8 proteins clearly demonstrate that ZIP8 is essential for the maintenance of normal Mn metabolism in the human body. The findings raise several questions regarding the essential role of ZIP8 in Mn metabolism. Why didn’t other metal transporters such as ZIP14 and DMT1, which are known to transport Mn, compensate for the deficiency of ZIP8? Was the undetectable level of blood Mn caused by the suppression of intestinal Mn uptake or by enhanced Mn excretion in bile or urine? What are the roles of the amino acids at the mutated sites in the permeation of Mn²⁺?

As to the intestinal Mn absorption, the roles of ZIP8 could be minimal because DMT1, which is highly expressed in the intestine and has a high affinity for Mn²⁺, can compensate for the absence of ZIP8.^23,24) On the other hand, biliary or urinary excretion of Mn could be affected by the defect in ZIP8. It has been known that a large part of Mn in the liver is excreted into the bile. A recent study which developed liver-specific ZIP8-knockout mice suggested that the lack of ZIP8 expression at the canalicular membrane of the bile duct might result in the reduced reabsorption of Mn in bile by the hepatic cells, leading to the depletion of Mn from the body²⁵⁾ (Fig. 1A). However, further studies are required to elucidate in more detail how biliary Mn is reabsorbed into hepatic cells by metal transporters including ZIP8. Since some of the biliary Mn could be reabsorbed from the intestine, the actual contribution of the defective reabsorption of biliary Mn to the drastic reduction of blood Mn levels in the ZIP8-mutated patients should be clarified.

Fig. 1. Schematic Description of Reabsorption of Mn via ZIP8 in the Bile Duct (A) and Proximal Tubule (B)

Another possibility is the enhanced urinary Mn excretion by the defect in ZIP8 function in the kidney. In an in vivo study, we demonstrated that ZIP8 is especially concentrated in the S3 segment of the mouse kidney proximal tubule, and in an in vitro study using the trans-well culture system, we showed that the suppression of ZIP8 resulted in reduced apical uptake of Mn²⁺ in immortalized PTECs.²¹⁾ These data suggest that ZIP8 may play an important role in the reabsorption of Mn from the proximal tubule lumen into the PTECs (Fig. 1B). DMT1 is also expressed in the kidney, but an immunohistochemical study showed that DMT1 is mainly located in the early endosomes within cells, where DMT1 may function primarily for the transport of endosomal iron derived from degraded transferrin-iron complexes to the cytosol.²⁶⁾ ZIP14 is also expressed in the kidneys, but whether ZIP14 can compensate for the lack of ZIP8 function in the reabsorption of luminal Mn remains to be elucidated.

A clinical study seems to support the role of renal ZIP8 in the maintenance of whole-body Mn level. A high dose of Mn given to the patients with ZIP8 mutations in Germany proved to be effective in curing symptoms of Mn deficiency. During the Mn therapy, the clinicians realized that urinary Mn excretion is much higher in such patients than in normal people though the variations in urinary Mn levels are very large.²⁷⁾ This notion suggests that a defect in renal ZIP8 function might have caused the reduced Mn reabsorption in the kidney, leading to increased urinary Mn excretion and consequently a decrease in the whole-body Mn level. Moreover, in contrast to the case in patients with ZIP8 mutations, the blood Mn levels in patients with SLC39A14 (ZIP 14) mutations were found to be higher than the normal level, suggesting that ZIP14 in the liver or kidney may not contribute to the reabsorption of Mn from the bile duct or proximal tubule, respectively.²⁸⁾

6. POSSIBLE ROLES OF MUTATED AMINO ACIDS IN ZIP8

The remaining important question regarding the mechanisms of Mn metabolism disorder caused by ZIP8 mutation is the roles of the amino acids in the mutated ZIP8 protein. This problem is difficult to elucidate due to the lack of knowledge regarding the 3-dimensional structure of mammalian ZIP8. Several hydrophobicity plots have provided models for predicting the transmembrane domains (TMDs) of ZIP8, but with inconsistent results. Recently, the X-ray-resolved crystal structure of bacterial ZIP4 was reported, and the structure of mammalian ZIP4 was predicted based on the similarity of amino acid sequences between bacterial and mammalian ZIP4.^29,30) Intriguingly, the result showed TMD structures different from those previously predicted from hydrophobicity plots. Since the sequences of amino acids covering TMD1 to TMD8 are somewhat homologous between ZIP4 and ZIP8, we present here a topological model of the TMD structure of ZIP8 based on the 3-dimensional structure of ZIP4 rather than the hydrophobicity plot (Fig. 2).

Fig. 2. Predicted Topology of Human ZIP8

Eight transmembrane domains (TMD) are indicated by Roman numbers. The mutated amino acids are located in the N-terminal extracellular domain (33, 38, and 112), TMD5 (335 and 340), and TMD7 (391).

The double mutations of ZIP8 in German patients were observed at the amino acid positions of 33 and 335 in one patient, and at 38 and 340 in another patient; in both the mutations were located in the N-terminal extracellular domain (33 and 38) and in TMD5 (335 and 340). In the case of ZIP4, it is predicted that TMD4 and TMD5 are the most important domains for the permeation of Zn, suggesting that the amino acid changes at the positions of 335 and 340 of ZIP8 may disturb the permeation of Mn in a similar way. The most prominent characteristic of the ZIP4 protein is its extraordinary long N-terminal extracellular domain. A recent study revealed that the N-terminal extracellular domain of ZIP4 is composed of multiple α-helix structures. Although the N-terminal extracellular domain of ZIP8 is not as long as that of ZIP4, the domain covering the amino acid positions of 33 and 38 shows similarities with one of the extracellular α-helixes of ZIP4.

Recently, a study reported that several recombinant ZIP8 proteins with mutations had been synthesized and expressed in HeLa cells.³¹⁾ The results showed that the mutated ZIP8 proteins, which have double mutations similar to those of German patients, exhibited a deficiency of Mn transport. The authors attributed the lost Mn transport ability to abnormal intracellular localization of the mutated ZIP8 in the endoplasmic reticulum rather than the plasma membrane.³¹⁾ However, this study solely showed the results of double mutations; the particular roles of amino acids at the positions of 33 and 38 in the N-terminal extracellular domain and those at 335 and 340, possibly located in TMD5, remain to be elucidated.

7. RELATIONSHIP BETWEEN ZIP8 SNPS AND DISEASES

In recent years, novel and unexpected findings regarding the relationship between human diseases and single nucleotide polymorphisms (SNPs) of genes have been provided by a variety of genome-wide association studies (GWASs). Several GWASs have shown the relationship between SNPs of the SLC39A8 gene and various diseases and symptoms such as schizophrenia,^32–34) hypertension,^35,36) obesity,^37,38) HDL cholesterol levels,^39–41) acute coronary syndrome,⁴²⁾ and Crohn’s disease.⁴³⁾ It is noteworthy that the same SNP (rs13107325) of the SLC39A8 gene that results in a change of amino acid (Ala391Thr) has been associated with all of these diseases and symptoms (Table 2). The amino acid position of 391 is located in the predicted TMD7 of ZIP8 (Fig. 2), but it remains unknown whether the change of alanine at this position to threonine alters the metal-transporting ability of ZIP8. On the other hand, no GWAS has so far detected the involvement of the SNPs causing missense mutations similar to those found in the glycosylation-deficient patients in Germany and Egypt, suggesting that the mutations found in these patients are very rare among the general population.

Table 2. Diseases and Metal Levels Related to ZIP8 SNPs Found in GWAS

Diseases symptoms	rs number	Mutations	References
Hypertension	rs13107325	c. 1171G > A (p. Ala391Thr)	Ehret et al., Nature (2011)
			Tragante et al., Am. H. Hum. Genet. (2014)
Obesity	rs13107325	c. 1171G > A (p. Ala391Thr)	Speliotes et al., Nat. Genet. (2010)
			Berndt et al., Nat. Genet. (2013)
HDL cholesterol	rs13107325	c. 1171G > A (p. Ala391Thr)	Teslovich et al., Nature (2010)
			Willer et al., Nat. Genet. (2013)
Schizophrenia	rs13107325	c. 1171G > A (p. Ala391Thr)	Hertzberg et al., Schizophr. Res. (2015)
			Ripke et al., Nature (2014)
			Costas, Am. J. Med. Genet. (2018)
Crohn’s disease	rs13107325	c. 1171G > A (p. Ala391Thr)	Li et al., Gastroenterology (2016)
Acute coronary syndrome	rs13107325	c. 1171G > A (p. Ala391Thr)	Johansson et al., Hum. Mol. Genet. (2016)
Metal levels
Blood Mn level	rs13107325	c. 1171G > A (p. Ala391Thr)	Ng et al., Hum. Mol. Genet. (2015)
Blood Zn level	rs233804	c. 840 + 12558G > T	Fujiwara et al., Leg. Med. (Tokyo) (2018)
		c. 639 + 12558G > T
Cd level in urine/erythrocyte	rs10014145/rs233804	c. 841 − 11341T > C	Rentschler et al., Metallomics (2014)
		c. 640 − 11341T > C

In addition, several studies have shown the relationship between the SNPs of ZIP8 and the levels of Mn, Zn and Cd in blood and urine^44–46) (Table 2). However, only the blood levels of Mn showed an association with the rs13107325 SNP.

The relation of a variety of diseases and symptoms with different etiologies to a single SNP of ZIP8 in GWAS raises important questions. Does the change of amino acid (Ala391Thr) affect the transporting ability of metals, especially Mn? If so, how are these multiple diseases and symptoms affected by disorders of Mn metabolism? Are the changes in Cd and Zn metabolism involved in all diseases or only particular symptoms?

Several kinds of explanations could be proposed as possible mechanisms. Changes in the activity of arginase, which is an Mn-dependent enzyme involved in nitric oxide (NO) production, could be related to blood pressure disorders.^47,48) Lipid abnormalities such as changes in HDL-cholesterol levels may be caused by the reduced glycosylation of many proteins due to a decline in the activity of Mn-dependent galactosyltransferase.^49,50) Disturbed metal deposition in the brain by altered ZIP8 function or immunological changes caused by ZIP8-regulated Zn metabolism may be possible causes for schizophrenia.⁵¹⁾ A study showed that the particular SNP (rs13107325) of ZIP8 is associated with alteration in the microbiome composition in the colon, which has been recently found to be associated with obesity, inflammatory diseases, lipid metabolism disorders, and schzophrenia.^43,52,53) However, as all these diseases and symptoms have polygenic and heterogenic causes, it may be difficult to provide an etiological explanation based on the dysfunction of a single metal transporter. Accumulation of further data and detailed biochemical analyses are required for elucidation of the relationship between ZIP8 dysfunction and multiple pathological conditions.

8. CONCLUSION

ZIP8 is a member of the Zn transporter family, but it plays important roles in the transport of toxic metal Cd and essential metal Mn. Recent findings in human studies have revealed that ZIP8 is deeply involved in the regulation of Mn homeostasis, and genetic variations of the SLC39A8 gene encoding human ZIP8 may be related to various diseases and symptoms. However, the precise mechanisms of ZIP8-regulated metabolisms of Mn and Cd remain obscure. Further studies on biochemical mechanisms of metal homeostasis regulated by ZIP8 are warranted.

Acknowledgments

This work was partly supported by JSPS KAKENHI Grant Number C-18K06646, the Vehicle Racing Commemorative Foundation, and the study of Health Effects of Heavy Metals organized by Ministry of the Environment, Japan.

Conflict of Interest

The authors declare no conflict of interest.

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