Protection of cultured vascular endothelial cells against cadmium cytotoxicity by simultaneous treatment or pretreatment with manganese

Abstract

Cadmium is a heavy metal that pollutes the environment and foods and is a risk factor for vascular disorders. We have previously demonstrated that pretreatment of vascular endothelial cells with zinc and copper protects the cells against cadmium cytotoxicity. In contrast, cadmium cytotoxicity was potentiated in cells following exposure to lead, thereby indicating that in vascular endothelial cells, cadmium cytotoxicity can be differentially modified by the co-occurrence of other heavy metals. In this study, we revealed that simultaneous treatment or pretreatment with manganese protects vascular endothelial cells against cadmium cytotoxicity. Intracellular accumulation of cadmium was observed to be reduced by simultaneous treatment with manganese, although not by pretreatment. The mRNA expression of metal transporters that regulate the uptake of both cadmium and manganese (ZIP8, ZIP14, and DMT1) remained unaffected by either simultaneous treatment or pretreatment with manganese, and simultaneous treatment with manganese suppressed the cadmium-induced expression of metallothionein but pretreatment with manganese did not exhibit such suppressive effect. Thus, the protection of vascular endothelial cells against cadmium cytotoxicity conferred by simultaneous treatment with manganese is assumed to be partially attributed to a reduction in the intracellular accumulation of cadmium, whereas the effects of pretreatment with manganese are independent of both the reduced intracellular accumulation of cadmium and the induction of metallothionein. These observations accordingly indicate that the protective effects of manganese are mediated via alternative (as yet unidentified) mechanisms.

INTRODUCTION

Vascular endothelial cells, which cover the luminal surface of blood vessels, play roles in regulating the blood coagulation–fibrinolytic system (Levin and Loskutoff, 1982; van Mourik et al., 1984) and vascular tone (Furchgott and Zawadzki, 1980; Yanagisawa et al., 1988). Given that vascular disorders, including atherosclerosis and hypertension, are triggered by endothelial abnormalities, it is important to clarify the molecular mechanisms underlying vascular endothelial cell dysfunction (Ross, 1999). As hazardous chemicals, including heavy metals, initially have to establish contact with blood vessels prior to entering parenchymal cells in target tissues, vascular endothelial cells represent a pivotal target for chemicals that exhibit toxicity against parenchymal cells (Kaji, 2004). For example, the vascular toxicity of methylmercury in the cerebral cortex results in neurotoxicity (Hirooka and Kaji, 2012; Yoshida et al., 2017).

Cadmium is a heavy metal and an environmental pollutant that occurs as a contaminant in foods such as rice. Cadmium is toxic to a diverse range of tissues, including the kidneys, bones, and liver (Dudley et al., 1985; Habeebu et al., 1998), whereas epidemiological studies have revealed that exposure to low-level cadmium is also a risk factor for atherosclerosis and hypertension (Revis et al., 1981; Houtman, 1993; Messner et al., 2009; Tellez-Plaza et al., 2012). The exhibition of cadmium toxicity generally depends on two factors, namely, the intracellular accumulation of cadmium and the induction of cellular defense systems. The former is associated with the activity of the ZIP transporters ZIP8 and ZIP14, which play roles in the uptake of cadmium into cells (Himeno et al., 2009), and we have found that the level of ZIP8 expression is an important factor in determining the sensitivity of vascular endothelial cells to cadmium toxicity (Fujie et al., 2022a). A further transporter, DMT1, mediates the import of divalent metal cations, including cadmium (Gunshin et al., 1997), and, collectively, these three transporters are associated with cadmium-induced cytotoxicity in vascular endothelial cells. Cellular defense against cadmium is primarily associated with the activity of metallothionein (MT), a cytoprotective protein that binds to cadmium and scavenges reactive oxygen species (Kägi, 1991; Sato and Bremner, 1993). In mammals, two inducible isoforms of MT, namely, MT-1 and MT-2, are ubiquitously expressed (Karin and Richards, 1982; Palmiter, 1994).

It has previously been established that cadmium-induced cytotoxicity in vascular endothelial cells is modified by the presence of other heavy metals such as zinc and copper, which play a protective role by reducing the intracellular accumulation of cadmium in these cells (Kaji et al., 1992a, 1992b, 1993), leading to reduction of cadmium-induced cytotoxicity independent of the induction of metallothionein (Kaji et al., 1992b). We revealed that lead as well as cadmium (Fujie et al., 2022a) induces ZIP8 in vascular endothelial cells (Fujie et al., 2022b). However, interaction of cadmium with other heavy metals remains to be determined.

Manganese, a trace element that binds to the active center of numerous enzymes, including superoxide dismutase, arginase, pyruvate carboxylase, and lactate dehydrogenase (Wedler, 1993), has similarly been observed to exhibit a protective effect against cadmium-induced cytotoxicity in cadmium-resistant MT-null cells (Himeno, 2002), although whether this metal plays a similar role in vascular endothelial cells has yet to be determined. In the present study, we accordingly sought to examine the interaction between cadmium and manganese in cultured vascular endothelial cells and characterize the associated mechanisms of action.

MATERIALS AND METHODS

Materials

Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the respective vendors: Dulbecco’s modified Eagle’s medium (DMEM) and calcium- and magnesium-free phosphate-buffered saline (CMF-PBS; Nissui Pharmaceutical, Tokyo, Japan); fetal bovine serum and a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA); QIAzol lysis reagent (QIAGEN, Valencia, CA, USA); GeneAce SYBR qPCR Mixα (Nippon Gene, Tokyo, Japan); mouse monoclonal anti-MT-1/2 antibody (E9; Dako, Glostrup, Denmark); Mouse monoclonal anti-β-actin antibody (Wako Pure Chemical Industries, Osaka, Japan); horseradish peroxidase-conjugated anti-mouse IgG antibody (#7076; Cell Signaling, Beverly, MA, USA); May-Grünwald and Giemsa stain solution (Merck KGaA, Darmstadt, Germany); and cadmium chloride, manganese chloride tetrahydrate, and other reagents (Nacalai Tesque, Kyoto, Japan).

Cell culture and treatment

Bovine aortic endothelial cells were cultured in a 5% CO₂ atmosphere at 37°C in DMEM supplemented with 10% fetal bovine serum until confluent. The medium was subsequently removed and the cells were washed twice with serum-free DMEM and exposed to cadmium chloride (1, 3, or 5 μM) or manganese chloride (5 or 10 μM) or both in serum-free DMEM for 6 or 24 hr, which we defined as the simultaneous treatment with manganese. For the pretreatment condition, the cells were washed twice with serum-free DMEM and then treated with manganese chloride (5 or 10 μM) in serum-free DMEM for 24 hr. The conditioning medium was then removed and the cells were exposed to cadmium chloride (1, 3, or 5 μM) in serum-free DMEM for 6 or 24 hr.

Giemsa staining

Confluent cultures of bovine aortic endothelial cells in 24-well plates were exposed to cadmium chloride (1, 3, or 5 μM) or manganese chloride (5 or 10 μM) or both in serum-free DMEM. Following incubation, the conditioning medium was harvested, and the cell layer was washed with CMF-PBS, fixed with methanol, and stained with Giemsa for morphological observations.

Cell viability assay

Confluent cultures of bovine aortic endothelial cells in 96-well plates were exposed to cadmium chloride (1, 3, or 5 μM) for 24 hr in serum-free DMEM under the condition of simultaneous treatment or pretreatment with manganese (5 or 10 μM). Following incubation, the conditioning medium was harvested, and the cell layer was incubated with 100 µL of fresh DMEM containing 0.25 mg/mL MTT for 4 hr. The DMEM was removed, and the cell layer was washed twice with CMF-PBS, and then lysed with 100 µL of dimethyl sulfoxide, after which the absorbance of the lysate at 570 nm was measured as a marker of cell viability using an ARVO Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). The cytotoxicity index (%) was calculated using the following formula:

where C_C is the viability of the cells treated without cadmium and manganese; Tc is that of the cells treated with cadmium and manganese.

Western blot analysis

Western blot analysis was performed as previously described (Fujie et al., 2016a). Briefly, confluent cultures of bovine aortic endothelial cells in 6-well plates were exposed to cadmium chloride (1, 3, or 5 μM) under the condition of simultaneous treatment or pretreatment with manganese (5 or 10 μM). After 24-hr incubation, the conditioning medium was removed, cells were washed twice with ice-cold CMF-PBS, lysed in 100 μL of sodium dodecyl sulfate sample buffer (50 mM Tris-HCl buffer solution containing 2% sodium dodecyl sulfate and 10% glycerol, pH 6.8), and incubated at 95°C for 5 min. The samples (10 µg protein) were mixed with 2-mercaptoethanol and bromophenol blue (1.67% each) and incubated at 95°C for 3 min. Total cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes, which were incubated with anti-MT-1/2 antibody (1:200) or anti-β-actin antibody (1:1000) at 4°C overnight, and subsequently incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody (1:5000) for 1 hr. Immunoreactive bands were visualized using enhanced chemiluminescence using Chemi-Lumi One Super and scanned using an Amersham imager 600 (GE Healthcare, Little Chalfont, UK).

Real-time RT-PCR

Confluent cultures of bovine aortic endothelial cells in 6-well plates were exposed to cadmium chloride (1, 3, or 5 μM) under the condition of simultaneous treatment or pretreatment with manganese (5 or 10 μM). After 6-hr incubation, the conditioned medium was discarded, and the cell layer was lysed with 300 μL of QIAzol lysis reagent. Total RNA was extracted from the lysate, and complementary DNA was synthesized using a high-capacity cDNA reverse transcription kit. Real-time PCR was performed using GeneAce SYBR qPCR Mixα with 10 ng cDNA and primers in a CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). The thermal cycling parameters were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The levels of ZIP8, ZIP14, DMT1, MT-1A, MT-2A, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs in each sample were quantified using the comparative Ct method. The fold change for each gene was assessed after normalization of the intensity value to that of GAPDH. The sequences of the forward and reverse primers used were as follows: bovine ZIP8, 5′- GAATGAGCACTCGACAAGCC-3′ (forward) and 5′- TAGAGGAACATGCCTCCAGC-3′ (reverse); bovine ZIP14, 5′-TCTCGGTAGTGCCTCTGTCC-3′ (forward) and 5′-GAATGTCTCAGTGCTGGTTGG-3′ (reverse); bovine DMT1, 5′- CACAGGTAGCCATCAGAGCC-3′ (forward) and 5′- ACCAGGTTAGGAGTTCAGGAG-3′ (reverse); bovine MT-1A, 5′-CACCTGCAAGGCCTGCAGA-3′ (forward) and 5′-CGAGGCCCCTTTGCAGACA-3′ (reverse); bovine MT-2A, 5′-GGCTCCTGCAAATGCAAAGAT-3′ (forward) and 5′-CCGAAGCCCCTTTGCAGAC-3′ (reverse); and bovine GAPDH, 5′-AACACCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-ACAGTCTTCTGGGTGGCAGTGA-3′ (reverse).

Intracellular accumulation of cadmium and manganese

Confluent cultures of bovine aortic endothelial cells in 6-well plates were exposed to cadmium chloride (1, 3, or 5 μM) under the condition of simultaneous treatment or pretreatment with manganese (5 or 10 μM). Following a 24-hr incubation, the conditioning medium was removed, cells were washed twice with ice-cold CMF-PBS, lysed in 100 μL of sodium dodecyl sulfate sample buffer (50 mM Tris-HCl buffer solution containing 2% sodium dodecyl sulfate and 10% glycerol, pH 6.8), and incubated at 95°C for 5 min. To degrade proteins, aliquots (50 μL) of the resulting lysate were treated with nitric acid at 130°C for 2 days. The resulting preparation was then dissolved in 5 mL of 0.1 M nitric acid and used for determinations of intracellular cadmium (m/z = 114) or manganese (m/z = 55) using inductively coupled plasma mass spectrometry (Nexion 300S; PerkinElmer, Waltham, MA, USA). Further aliquots of the lysates (30 μL) were analyzed for DNA content using a fluorometric method (Kissane and Robins, 1958) according to the expression of cadmium or manganese (pmol/μg DNA).

Statistical analysis

Data were analyzed for statistical significance using an analysis of variance (ANOVA) and Bonferroni/Dunn t-test, when possible. P values of less than 0.05 were considered to be statistically significant.

RESULTS

First, we investigated the effect of manganese on cadmium-induced cytotoxicity in vascular endothelial cells based on morphological observation and cell viability assay. As monolayers, vascular endothelial cells have a cobblestone-like appearance, and monolayers exposed to cadmium tend to be characterized by occurrence of areas of de-endothelialization (Kaji et al., 1992c). Consistently, we found that exposure to cadmium resulted in de-endothelialization of the vascular endothelial cell layer, which was not observed in cells treated with manganese alone (Fig. 1A). In contrast, however, we detected no evidence of cadmium-induced de-endothelialization in cells simultaneously treated with manganese. The cell viability of vascular endothelial cells exposed to cadmium was decreased, which was recovered by the simultaneous exposure to manganese (Fig. 1B), thereby indicating that simultaneous exposure to manganese protects vascular endothelial cells against cadmium-induced cytotoxicity. Similarly, we detected no cadmium-induced de-endothelialization following pretreatment with manganese (Fig. 1C), and the cadmium-decreased cell viability was recovered by the pretreatment with manganese (Fig. 1D). Accordingly, both pretreatment and simultaneous treatment with manganese appear to protect vascular endothelial cells against the effects of cadmium-induced cytotoxicity.

Fig. 1

Influence of manganese on cadmium-induced cytotoxicity in vascular endothelial cells. [A and C] Morphological appearance of vascular endothelial cells following exposure to cadmium and manganese. Vascular endothelial cells were exposed [A] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 24 hr or [C] to cadmium (1, 3, or 5 µM) for 24 hr subsequent to pretreatment with manganese (5 or 10 µM) for 24 hr. The cell layer was stained using Giemsa stain. Original magnification (×40). [B and D] Vascular endothelial cells were exposed [B] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 24 hr or [D] to cadmium (1, 3, or 5 µM) for 24 hr subsequent to pretreatment with manganese (5 or 10 µM) for 24 hr. The cytotoxicity index was represented by cell viability measured by MTT assay. Values are the means ± SE of four independent samples. Statistical significance when compared with the corresponding cadmium-exposed cells without manganese, *p < 0.05, **p < 0.01.

We subsequently examined the intracellular accumulation of cadmium and manganese in vascular endothelial cells and observed that the increase in intracellular accumulation of cadmium in vascular endothelial cells exposed to cadmium was suppressed by the simultaneous treatment with manganese (Fig. 2A, left panel). In response to treatment with manganese, we observed an increase in the accumulation of this metal (Fig. 2A, right panel), although no significant change in accumulation was detected when cells were simultaneously exposed to cadmium, thus indicating that increases in the intracellular accumulation of cadmium in vascular endothelial cells exposed to cadmium were suppressed by the simultaneous treatment with manganese. In contrast, pretreatment with manganese had no comparable suppressive effect on the intracellular accumulation of cadmium (Fig. 2B, left panel), indicating that pretreatment with manganese had no marked influence on the intracellular accumulation of cadmium in vascular endothelial cells following exposure to cadmium. Like cadmium, significant change in the intracellular accumulation of manganese was not observed by pretreatment with manganese (Fig. 2B, right panel).

Fig. 2

Intracellular accumulation of cadmium and manganese in vascular endothelial cells. Vascular endothelial cells were exposed [A] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 24 hr or [B] to cadmium (1, 3, or 5 µM) for 24 hr following pretreatment with manganese (5 or 10 µM) for 24 hr, and the intracellular accumulation of cadmium and manganese was measured by inductively coupled plasma mass spectrometry. Values are the means ± SE of four independent samples. Statistical significance when compared with the corresponding cadmium-exposed cells without manganese, *p < 0.05, **p < 0.01.

Analysis of the expression levels of metal transporter mRNAs revealed increases in the levels of ZIP8 and ZIP14 mRNAs, but reductions in those of DMT1 mRNA in response to cadmium exposure (Fig. 3A), and the induction of ZIP8 and ZIP14 by cadmium is supported by our previous study (Fujie et al., 2022a). However, neither simultaneous treatment nor pretreatment with manganese had any significant effects on the levels of these transporter mRNAs, and we observed no significant reduction in the cadmium-induced elevation of ZIP8 and ZIP14 mRNA expression in response to either simultaneous treatment or pretreatment with manganese, indicating that manganese probably has no marked influence on the expression of the three assessed transporters.

Fig. 3

Levels of ZIP8, ZIP14, and DMT1 mRNAs in vascular endothelial cells following exposure to cadmium and manganese. Vascular endothelial cells were exposed [A] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 6 hr or [B] to cadmium (1, 3, or 5 µM) for 6 hr following pretreatment with manganese (5 or 10 µM) for 24 hr, and the levels of ZIP8, ZIP14, and DMT1 mRNAs were determined using real-time RT-PCR. Values are the means ± SE of three technical replicates. Statistical significance when compared with the corresponding cells without cadmium and manganese, *p < 0.05, **p < 0.01.

Figure 4 shows the induction of MT in vascular endothelial cells in response to exposure to cadmium or manganese or both. We observed increases in the expression of MT-1/2 proteins in vascular endothelial cells treated with cadmium, which were partially suppressed by the simultaneous treatment with manganese (Fig. 4A). We then proceeded to investigate the effects of manganese on cadmium-mediated transcriptional induction of the main isoforms of MT (MT-1A and MT-2A) expressed in bovine cells (Fujie et al., 2020). As shown in Fig. 4B, there were increases in levels of MT-1A and MT-2A mRNAs in vascular endothelial cells following exposure to cadmium, which were partially suppressed by the simultaneous treatment with manganese. Contrastingly, pretreatment with manganese appeared to have no similar suppressive effect on cadmium-induced MT-1/2 protein expression (Fig. 4C). Additionally, we found that pretreatment with manganese had no significant suppressive effect on the cadmium-induced upregulation of endothelial MT-1A and MT-2A mRNAs (Fig. 4D), indicating that the pretreatment did not affect the induction of endothelial MT expression by cadmium.

Fig. 4

Induction of metallothionein by cadmium and manganese in vascular endothelial cells. Vascular endothelial cells were exposed [A] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 24 hr or [C] to cadmium (1, 3, or 5 µM) for 24 hr following pretreatment with manganese (5 or 10 µM) for 24 hr, and the expression of MT-1/2 proteins was determined using western blot analysis. The ratio of the intensity of MT-1/2 in Fig. 4A or 4C to those of β-actin; values are the means of two independent samples. [B and D] Levels of metallothionein gene expression in vascular endothelial cells treated with cadmium and manganese. Vascular endothelial cells were exposed [B] to cadmium (1, 3, or 5 µM) and manganese (5 or 10 µM) for 6 hr or [D] to cadmium (1, 3, or 5 µM) for 6 hr following pretreatment with manganese (5 or 10 µM) for 24 hr, and the levels of MT-1A and MT-2A mRNAs were determined using real-time RT-PCR. Values are the means ± SE of three technical replicates. Statistical significance when compared with the corresponding cadmium-exposed cells without manganese, *p < 0.05, **p < 0.01.

DISCUSSION

In this study, we obtained the following results: (1) Both simultaneous treatment and pretreatment with manganese contributed to protecting vascular endothelial cells against cadmium-induced cytotoxicity. (2) Although an increase in the intracellular accumulation of cadmium was suppressed by simultaneous treatment with manganese, no comparable effect was observed in response to pretreatment with manganese. (3) Neither simultaneous treatment nor pretreatment with manganese influenced mRNA expression of the transporters ZIP8, ZIP14, and DMT1. (4) Simultaneous treatment with manganese, although not pretreatment, partially suppressed the cadmium-induced expression of endothelial MT. Collectively, our findings indicate that manganese protects vascular endothelial cells against cadmium-induced cytotoxicity, and that the mechanisms underlying the protective effect of simultaneous treatment with manganese are partially associated with a reduction in the intracellular accumulation of cadmium. In contrast, however, neither the intracellular accumulation of cadmium nor the cadmium-induced expression of endothelial MT appeared to be affected by pretreatment with manganese, thereby indicating that pretreatment with manganese protects cells against cadmium-induced cytotoxicity via alternative mechanisms.

ZIP8 and ZIP14, members of the ZIP transporter family, are mainly localized in the plasma membrane (Grotz et al., 1998; Fukada and Kambe, 2011). Given their high degree of homology, they are assumed to have similar functions regarding the transport of zinc, iron, cadmium, and manganese from the extracellular space into the cell cytosol (Girijashanker et al., 2008). DMT1 plays comparable roles in the transport of divalent metal cations, including zinc, copper, iron, manganese, cobalt, nickel, cadmium, and lead (Gunshin et al., 1997). In the present study, we found that manganese treatment had no significant effect on the levels of ZIP8, ZIP14, or DMT1 mRNA in vascular endothelial cells regardless of cadmium exposure. Our observations indicating that the intracellular accumulation of cadmium in vascular endothelial cells is suppressed by simultaneous treatment with manganese could, therefore, be indicative of competition between cadmium and manganese for uptake into the cell via these common transporters. However, it is conceivable that the mechanisms underlying the suppression of cadmium accumulation by simultaneous treatment with manganese may differ from those pertaining to zinc (Kaji et al., 1992b) and copper (Kaji et al., 1992a), given that these two metals have been shown to have a higher affinity for mouse ZIP8 protein than cadmium, whereas manganese has a lower affinity (Koike et al., 2017).

MT-1 and MT-2, which are induced in response to heavy metal exposure, play pivotal cytoprotective roles against cadmium toxicity. The induction of these proteins requires the initial activation of the metal response element (MRE) in the promoter region of the MT gene (Bittel et al., 1998). MRE-binding transcription factor-1 (MTF-1) is a transcription factor that binds to the MRE, a process that is dependent on the presence of zinc ions (Radtke et al., 1993; Heuchel et al., 1994; Zhang et al., 2001). However, although zinc has been characterized as a typical inducer of MT-1 and MT-2, it appears that endothelial MT is not induced by this metal (Kaji et al., 1992b; Fujie et al., 2016a). In this regard, we have previously shown that in vascular endothelial cells, the cadmium-mediated induction of MT is regulated not only by the MTF-1-MRE pathway but also by the Nrf2-ARE pathway (Shinkai et al., 2016). Moreover, differences in the mechanisms of MT-1 and MT-2 induction in vascular endothelial cells have been reported using copper diethyldithiocarbamate and tris(pentafluorophenyl)stibane (Fujie et al., 2016b, 2016c). In this study, we found that cadmium-induced endothelial MT was suppressed by simultaneous treatment with manganese and that the suppression of the MT-1A gene was more pronounced than that of MT-2A gene, thereby indicating that the suppression of cadmium toxicity attributed to the reduced intracellular accumulation of cadmium by simultaneous treatment with manganese, and that the expression of MT-1 is suppressed to a greater extent by manganese than is that of MT-2.

Our findings in the present study revealed that both simultaneous treatment and pretreatment with manganese protected vascular endothelial cells against cadmium-induced cytotoxicity. In case of simultaneous treatment with manganese, the mechanisms were found to be independent of the induction of endothelial MT and partially due to a reduction in cadmium accumulation by competition between cadmium and manganese for uptake into the cell via common metal transporters (ZIP8, ZIP14, and DMT1). We reported that zinc protects various cell types other than vascular endothelial cells through metallothionein-independent mechanisms as well as metallothionein induction (Mishima et al., 1996; Mishima et al., 1997). Interaction of cadmium with manganese would be similar to that with zinc. Additionally, it is indicated that the observed responses in the cells pretreated with manganese are also attributable to other (as yet to be determined) mechanisms, which could be the mechanisms characteristic or specific to vascular endothelial cells. Further studies are accordingly required to clarify the mechanisms associated with the protective effects of manganese on vascular endothelial cells.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Numbers JP22K17355 (to T. H.), JP23K16312 (to T. F.), and the Study Group of the Health Effects of Heavy Metals Organized by the Ministry of the Environment, Japan. We would like to thank Editage (www.editage.com) for English language editing.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Bittel,  D.,  Dalton,  T.,  Samson,  S.L.,  Gedamu,  L. and  Andrews,  G.K. (1998): The DNA binding activity of metal response element-binding transcription factor-1 is activated in vivo and in vitro by zinc, but not by other transition metals. J. Biol. Chem., 273, 7127-7133.
•  Dudley,  R.E.,  Gammal,  L.M. and  Klaassen,  C.D. (1985): Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. Appl. Pharmacol., 77, 414-426.
•  Fujie,  T.,  Ito,  K.,  Ozaki,  Y.,  Takahashi,  S.,  Yamamoto,  C. and  Kaji,  T. (2022a): Induction of ZIP8, a ZIP transporter, via NF-κB signaling by the activation of IκBα and JNK signaling in cultured vascular endothelial cells exposed to cadmium. Toxicol. Appl. Pharmacol., 434, 115802.
•  Fujie,  T.,  Murakami,  M.,  Yoshida,  E.,  Yasuike,  S.,  Kimura,  T.,  Fujiwara,  Y.,  Yamamoto,  C. and  Kaji,  T. (2016c): Transcriptional induction of metallothionein by tris(pentafluorophenyl)stibane in cultured bovine aortic endothelial cells. Int. J. Mol. Sci., 17, 1381.
•  Fujie,  T.,  Ozaki,  Y.,  Takenaka,  F.,  Nishio,  M.,  Hara,  T.,  Fujiwara,  Y.,  Yamamoto,  C. and  Kaji,  T. (2020): Induction of metallothionein isoforms in cultured bovine aortic endothelial cells exposed to cadmium. J. Toxicol. Sci., 45, 801-806.
•  Fujie,  T.,  Muraoka,  A.,  Ito,  K.,  Ozaki,  Y.,  Yamamoto,  C. and  Kaji,  T. (2022b): Lead induces a metal transporter, ZIP8, via activation of the NF-κB signaling pathway and the induction is involved in the protection against lead cytotoxicity by intracellular lead accumulation independent mechanisms in cultured vascular endothelial cells. J. Toxicol. Sci., 47, 493-501.
•  Fujie,  T.,  Segawa,  Y.,  Uehara,  A.,  Nakamura,  T.,  Kimura,  T.,  Yoshida,  E.,  Yamamoto,  C.,  Uchiyama,  M.,  Naka,  H. and  Kaji,  T. (2016a): Zinc diethyldithiocarbamate as an inducer of metallothionein in cultured vascular endothelial cells. J. Toxicol. Sci., 41, 217-224.
•  Fujie,  T.,  Segawa,  Y.,  Yoshida,  E.,  Kimura,  T.,  Fujiwara,  Y.,  Yamamoto,  C.,  Satoh,  M.,  Naka,  H. and  Kaji,  T. (2016b): Induction of metallothionein isoforms by copper diethyldithiocarbamate in cultured vascular endothelial cells. J. Toxicol. Sci., 41, 225-232.
•  Fukada,  T. and  Kambe,  T. (2011): Molecular and genetic features of zinc transporters in physiology and pathogenesis. Metallomics, 3, 662-674.
•  Furchgott,  R.F. and  Zawadzki,  J.V. (1980): The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373-376.
•  Girijashanker,  K.,  He,  L.,  Soleimani,  M.,  Reed,  J.M.,  Li,  H.,  Liu,  Z.,  Wang,  B.,  Dalton,  T.P. and  Nebert,  D.W. (2008): Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter. Mol. Pharmacol., 73, 1413-1423.
•  Grotz,  N.,  Fox,  T.,  Connolly,  E.,  Park,  W.,  Guerinot,  M.L. and  Eide,  D. (1998): Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc. Natl. Acad. Sci. USA, 95, 7220-7224.
•  Gunshin,  H.,  Mackenzie,  B.,  Berger,  U.V.,  Gunshin,  Y.,  Romero,  M.F.,  Boron,  W.F.,  Nussberger,  S.,  Gollan,  J.L. and  Hediger,  M.A. (1997): Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature, 388, 482-488.
•  Habeebu,  S.S.,  Liu,  J. and  Klaassen,  C.D. (1998): Cadmium-induced apoptosis in mouse liver. Toxicol. Appl. Pharmacol., 149, 203-209.
•  Heuchel,  R.,  Radtke,  F.,  Georgiev,  O.,  Stark,  G.,  Aguet,  M. and  Schaffner,  W. (1994): The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J., 13, 2870-2875.
•  Himeno,  S. (2002): Application of metallothionein null cells to investigation of cadmium transport. J. Inorg. Biochem., 88, 207-212.
•  Himeno,  S.,  Yanagiya,  T. and  Fujishiro,  H. (2009): The role of zinc transporters in cadmium and manganese transport in mammalian cells. Biochimie, 91, 1218-1222.
•  Hirooka,  T. and  Kaji,  T. (2012): The cytotoxicity of methylmercury in human microvascular endothelial cells and pericytes in culture. Biol. Pharm. Bull., 35, 1201-1205.
•  Houtman,  J.P. (1993): Prolonged low-level cadmium intake and atherosclerosis. Sci. Total Environ., 138, 31-36.
•  Karin,  M. and  Richards,  R.I. (1982): Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature, 299, 797-802.
•  Kägi,  J.H. (1991): Overview of metallothionein. Methods Enzymol., 205, 613-626.
•  Kaji,  T. (2004): Cell biology of heavy metal toxicity in vascular tissue. Yakugaku Zasshi, 124, 113-120.
•  Kaji,  T.,  Fujiwara,  Y.,  Koyanagi,  E.,  Yamamoto,  C.,  Mishima,  A.,  Sakamoto,  M. and  Kozuka,  H. (1992a): Protective effect of copper against cadmium cytotoxicity on cultured vascular endothelial cells. Toxicol. Lett., 63, 13-20.
•  Kaji,  T.,  Mishima,  A.,  Koyanagi,  E.,  Yamamoto,  C.,  Sakamoto,  M. and  Kozuka,  H. (1992b): Possible mechanism for zinc protection against cadmium cytotoxicity in cultured vascular endothelial cells. Toxicology, 76, 257-270.
•  Kaji,  T.,  Mishima,  A.,  Yamamoto,  C.,  Sakamoto,  M. and  Koizumi,  F. (1992c): Effect of cadmium on the monolayer maintenance of vascular endothelial cells in culture. Toxicology, 71, 267-276.
•  Kaji,  T.,  Mishima,  A.,  Yamamoto,  C.,  Sakamoto,  M. and  Kozuka,  H. (1993): Zinc protection against cadmium-induced destruction of the monolayer of cultured vascular endothelial cells. Toxicol. Lett., 66, 247-255.
•  Kissane,  J.M. and  Robins,  E. (1958): The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chem., 233, 184-188.
•  Koike,  A.,  Sou,  J.,  Ohishi,  A.,  Nishida,  K. and  Nagasawa,  K. (2017): Inhibitory effect of divalent metal cations on zinc uptake via mouse Zrt-/Irt-like protein 8 (ZIP8). Life Sci., 173, 80-85.
•  Levin,  E.G. and  Loskutoff,  D.J. (1982): Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J. Cell Biol., 94, 631-636.
•  Messner,  B.,  Knoflach,  M.,  Seubert,  A.,  Ritsch,  A.,  Pfaller,  K.,  Henderson,  B.,  Shen,  Y.H.,  Zeller,  I.,  Willeit,  J.,  Laufer,  G.,  Wick,  G.,  Kiechl,  S. and  Bernhard,  D. (2009): Cadmium is a novel and independent risk factor for early atherosclerosis mechanisms and in vivo relevance. Arterioscler. Thromb. Vasc. Biol., 29, 1392-1398.
•  Mishima,  A.,  Yamamoto,  C.,  Fujiwara,  Y. and  Kaji,  T. (1996): Metallothionein-independent protection by low levels of zinc against cadmium cytotoxicity in cultured cells. Res. Commun. Pharmacol. Toxicol., 1, 199-210.
•  Mishima,  A.,  Yamamoto,  C.,  Fujiwara,  Y. and  Kaji,  T. (1997): Tolerance to cadmium cytotoxicity is induced by zinc through non-metallothionein mechanisms as well as metallothionein induction in cultured cells. Toxicology, 118, 85-92.
•  van Mourik,  J.A.,  Lawrence,  D.A. and  Loskutoff,  D.J. (1984): Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. J. Biol. Chem., 259, 14914-14921.
•  Palmiter,  R.D. (1994): Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc. Natl. Acad. Sci. USA, 91, 1219-1223.
•  Radtke,  F.,  Heuchel,  R.,  Georgiev,  O.,  Hergersberg,  M.,  Gariglio,  M.,  Dembic,  Z. and  Schaffner,  W. (1993): Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J., 12, 1355-1362.
•  Revis,  N.W.,  Zinsmeister,  A.R. and  Bull,  R. (1981): Atherosclerosis and hypertension induction by lead and cadmium ions: an effect prevented by calcium ion. Proc. Natl. Acad. Sci. USA, 78, 6494-6498.
•  Ross,  R. (1999): Atherosclerosis--an inflammatory disease. N. Engl. J. Med., 340, 115-126.
•  Sato,  M. and  Bremner,  I. (1993): Oxygen free radicals and metallothionein. Free Radic. Biol. Med., 14, 325-337.
•  Shinkai,  Y.,  Kimura,  T.,  Itagaki,  A.,  Yamamoto,  C.,  Taguchi,  K.,  Yamamoto,  M.,  Kumagai,  Y. and  Kaji,  T. (2016): Partial contribution of the Keap1-Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells. Toxicol. Appl. Pharmacol., 295, 37-46.
•  Tellez-Plaza,  M.,  Navas-Acien,  A.,  Menke,  A.,  Crainiceanu,  C.M.,  Pastor-Barriuso,  R. and  Guallar,  E. (2012): Cadmium exposure and all-cause and cardiovascular mortality in the U.S. general population. Environ. Health Perspect., 120, 1017-1022.
•  Wedler,  F.C. (1993): Biological significance of manganese in mammalian systems. Prog. Med. Chem., 30, 89-133.
•  Yanagisawa,  M.,  Kurihara,  H.,  Kimura,  S.,  Tomobe,  Y.,  Kobayashi,  M.,  Mitsui,  Y.,  Yazaki,  Y.,  Goto,  K. and  Masaki,  T. (1988): A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415.
•  Yoshida,  E.,  Kurita,  M.,  Eto,  K.,  Kumagai,  Y. and  Kaji,  T. (2017): Methylmercury promotes prostacyclin release from cultured human brain microvascular endothelial cells via induction of cyclooxygenase-2 through activation of the EGFR-p38 MAPK pathway by inhibiting protein tyrosine phosphatase 1B activity. Toxicology, 392, 40-46.
•  Zhang,  B.,  Egli,  D.,  Georgiev,  O. and  Schaffner,  W. (2001): The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Mol. Cell. Biol., 21, 4505-4514.