Zinc Supplementation Protects against Cadmium Accumulation and Cytotoxicity in Madin-Darby Bovine Kidney Cells

Abstract

Cadmium ions (Cd²⁺) have been reported to accumulate in bovine tissues, although Cd²⁺ cytotoxicity has not been investigated thoroughly in this species. Zinc ions (Zn²⁺) have been shown to antagonize the toxic effects of heavy metals such as Cd²⁺ in some systems. The present study investigated Cd²⁺ cytotoxicity in Madin-Darby bovine kidney (MDBK) epithelial cells, and explored whether this was modified by Zn²⁺. Exposure to Cd²⁺ led to a dose- and time-dependent increase in apoptotic cell death, with increased intracellular levels of reactive oxygen species and mitochondrial damage. Zn²⁺ supplementation alleviated Cd²⁺-induced cytotoxicity and this protective effect was more obvious when cells were exposed to a lower concentration of Cd²⁺ (10 μM), as compared to 50 μM Cd²⁺. This indicated that high levels of Cd²⁺ accumulation might induce irreversible damage in bovine kidney cells. Metallothioneins (MTs) are metal-binding proteins that play an essential role in heavy metal ion detoxification. We found that co-exposure to Zn²⁺ and Cd²⁺ synergistically enhanced RNA and protein expression of MT-1, MT-2, and the metal-regulatory transcription factor 1 in MDBK cells. Notably, addition of Zn²⁺ reduced the amounts of cytosolic Cd²⁺ detected following MDBK exposure to 10 μM Cd²⁺. These findings revealed a protective role of Zn²⁺ in counteracting Cd²⁺ uptake and toxicity in MDBK cells, indicating that this approach may provide a means to protect livestock from excessive Cd²⁺ accumulation.

Introduction

Cadmium (Cd) is a heavy metal that is extensively used in the manufacture of alloys, pigments, electroplates, and batteries. The toxic effects of free cadmium ions (Cd²⁺) have been studied intensively in humans, and effects on a wide range of organs have been reported, including the liver, bones, kidneys, and the reproductive, neurological, and immunological system [1], [2]. Acute Cd²⁺ toxicity in the respiratory and digestive systems causes severe chemical pneumonitis and bloody diarrhea, respectively [3]. However, the kidney and skeleton are most affected by chronic Cd²⁺ toxicity. With chronic exposure, around 50% of the absorbed Cd²⁺ accumulates in the kidneys, and syndromes associated with Cd²⁺-induced renal damage include impaired vitamin metabolism, proteinuria, and loss of bone calcium [4]. Even though Cd exposure has traditionally been thought to occur in industrializing developing counties because of environmental pollution, it is causing growing concern worldwide because Cd²⁺ can accumulate over time in animals and plants used in human food products [5]. For example, Cd²⁺ accumulation to levels high enough to cause toxic effects in humans was reported in a polish study of cattle in 1999 [6].

Following its absorption into cells, Cd²⁺ complexes with members of the metallothionein (MT) family of conserved low-molecular-weight cysteine- and metal-rich proteins. In mammals, MTs exist mainly in the cytoplasm, but can also be detected in lysosomes, mitochondria, and nuclei. Four MT isoforms, designated MT-1 to MT-4, have been identified. MT-1 and MT-2 are the predominant isoforms and are expressed in most tissues, whereas MT-3 and MT-4 are constitutively expressed in the central nervous system and the stratified squamous epithelium, respectively [7]. A wide range of metals rapidly induce MT-1 and MT-2 transcription via metal-regulatory transcription factor 1 (MTF-1) binding to the metal-responsive elements (MREs) within their promoter regions [8]. In addition, cellular stressors, hormones, reactive oxygen species (ROS), and cytokines can also affect MT gene transcription [9]. MTs play an essential role in the homeostasis of essential metal ions, in addition to the sequestration and detoxification of Cd²⁺ and other heavy metals. Furthermore, MTs are efficient scavengers of free radicals generated during oxidative stress [10]. Free Cd²⁺ levels can increase owing to either excessive exposure to Cd²⁺ or MT deficiency, and this can lead to a wide variety of cytotoxic effects.

In humans, Cd²⁺ induces apoptosis via both caspase-dependent and -independent pathways [11]. Caspases are aspartate-specific cysteine proteases that trigger proteolytic cascades and induce amplification of intracellular apoptotic signals. In human kidney proximal tubule cells, Cd²⁺ was found to induce activation of caspase-9 and caspase-3, probably via the release of cytochrome c from damaged mitochondria [12]. Caspase-independent apoptosis can occur by Cd²⁺-mediated effects on the tumor suppressor protein (p53), because Cd²⁺ can replace Zn²⁺ within p53 and thereby compromise p53-mediated DNA damage repair or cell cycle arrest [11]. Cd²⁺ can also activate the Ca²⁺-dependent protease, calpain, which plays an essential role in Cd²⁺-induced caspase-independent apoptosis at early time points in rat kidney proximal tubule cells [12]. Cd²⁺-induced apoptosis is associated with ROS accumulation, which can induce mitochondrial, DNA, and protein damage [13].

Zinc (Zn) is an essential trace element that plays a pivotal role in the structural stability of Zn²⁺-dependent proteins and in antagonizing the toxic effects of heavy metals such as Cd²⁺ [14], [15], although exposure to Zn²⁺ can also accelerate apoptosis [16]. Zn²⁺ supplementation counteracted acute Cd²⁺-induced nephrotoxicity in a mouse model [17] and the amounts of Cd²⁺ identified in cattle were inversely proportional to the levels of Zn²⁺ [6]. However, little is known about Cd²⁺-mediated toxicity in cows, or the protective effects of Zn²⁺ supplementation in this animal. This issue has direct economic relevance to food production and important indirect consequences for global public health. The objectives of this study were therefore to investigate Cd²⁺ toxicity in bovine kidney cells and to explore whether Zn²⁺ supplementation prevented Cd²⁺ absorption and/or cytotoxicity.

Materials and Methods

Cell culture and treatment

The Madin-Darby bovine kidney epithelial cell line (MDBK, obtained from Boster, Wuhan, China) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded into a 6-well cell culture plate and grown to approximately 80% confluence before exposure to CdCl₂ alone, or in combination with ZnCl₂, for the indicated time periods. Control cells were treated with growth medium (RPMI with 10% FBS).

Measurement of cell damage

The number of cells undergoing apoptosis and necrosis was determined by flow cytometry after staining with fluorescein isothiocyanate (FITC)-conjugated annexin V antibody and propidium iodide (PI) according to the manufacturer's instructions. Data were acquired by FACS Calibur (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo 7.6.4 software (Tree Star Inc., Ashland, OR, USA).

RNA isolation and real-time polymerase chain reaction (PCR)

Total RNA was extracted from cells using the TRI Reagent (Invitrogen, USA) and reverse transcribed using a cDNA Synthesis Kit (DingGuo, TER016-2, Wuhan, China). Real-time PCR was performed on a 7500 real-time PCR system (Applied Biosystems, USA) using SYBR-Green. mt-1, mt-2, mtf-1, and glyceraldehyde 3-phosphate dehydrogenase (gapdh) were amplified using the primers shown in Table S1. Each assay was carried out in a 20-μl reaction mixture containing 50 ng of cDNA, 0.2 μM of each primer, 10 μl of reaction mix, and 0.5 μl of SYBR-Green dye (TaKaRa, Dalian, China). The thermal cycling conditions included an initial denaturation step of 94°C for 4 min, followed by 40 amplification cycles of 92°C for 1 min, 60°C for 1 min, and 72°C for 1 min. Threshold cycle (Ct) values of the target genes were normalized to that of the reference gene (GAPDH) and expressed as fold changes, compared to those associated with the control sample.

Immunofluorescence staining

Cells were fixed with 4% neutral formaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 20 min; unspecific binding sites were blocked with 10% bovine serum albumin (BSA) for 30 min before immunofluorescence staining. Cells were incubated with a mouse anti-MT antibody (ab12228, 1∶150, Abcam, Hong Kong, China) that reacts with both MT1 and MT2 or a rabbit anti-MTF-1 antibody (1∶200, EIAab, Wuhan, China) overnight at 4°C, followed by incubation with FITC- or Texas red-conjugated secondary antibodies (1∶100, Proteintech Group Inc., Wuhan, China) for 30 min. Controls for unspecific binding of antibodies were included in each staining. Images of cells were captured using a confocal fluorescence microscope (NOL-LSM710, Carl Zeiss Jena, Germany) and analyzed using ZEN 2009 light edition software.

Western blot analysis

Cells were lysed in 50 μl of lysis buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, and 0.1 mM SDS). The protein concentration was determined and equal amounts of protein were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride membranes, and these were incubated with a mouse anti-MT antibody (1∶400, Abcam, Hong Kong, China) or a rabbit anti-MTF-1 antibody (1∶500, EIAab, Wuhan, China). Biotinylated goat anti-mouse or goat anti-rabbit IgG (1∶8000, Boster Company, Wuhan, China) was used as the secondary antibody. Signals were detected using an enhanced chemiluminescence kit (Beyotime, Shanghai, China).

Measurement of intracellular ROS

Levels of intracellular ROS were detected using a method reported previously [18]. Briefly, treated cells were incubated with 10 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probes suspended in serum-free medium at 37°C for 30 min. After washing with phosphate-buffered saline (PBS) three times, ROS were monitored by following the intracellular conversion of DCFH-DA into the fluorescent product, dichlorofluorescein (DCF), using either fluorescence microscopy or flow cytometry.

Measurement of the mitochondrial membrane potential

The mitochondrial membrane potential (MMP) was measured using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), a cationic lipophilic fluorescent dye that can selectively enter mitochondria and reflect MMP through reversible color changes. JC-1 forms complexes and produced red fluorescence in healthy cells exhibiting high MMP; in apoptotic cells with low MMP, JC-1 remains in a monomeric form and emits green fluorescence. In brief, treated cells were incubated with 5 μg/ml of JC-1 for 20 min at 37°C. After intensive washing, the relative amounts of mitochondrial red and green fluorescence were determined by either flow cytometry or fluorescence microscope.

Quantification of intracellular Cd²⁺ and Zn²⁺

Levels of intracellular Cd²⁺ and Zn²⁺ were quantified by quadrupole inductively coupled plasma mass spectrometry (ICP-MS). Cells were intensively washed with PBS, quantified, and transferred to acid-washed high-density polyethylene bottles. Cells were digested with HNO₃/HCl (1∶3) at room temperature for 1 day and then completely evaporated by heating. The sediments were re-dissolved with 0.1% HNO₃ to a volume of 6 ml. Metals were measured using the following operating conditions: 0.8 L/min gas box nebulizer flow, 0.6 L/min gas box auxiliary flow, 10 s acquisition time, three replicates, and 2400 V radio frequency power.

Statistical analysis

All experiments were repeated at least three times. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 17.0 software (SPSS Inc., IL, USA). All data are expressed as the mean ± standard deviation (SD) of one representative experiment, performed in quadruplicate. Paired Student's t-tests were used to analyze the effect of Cd²⁺- or Zn²⁺-exposure, where P<0.05 was considered statistically significant.

Results

Effects of Zn²⁺ and Cd²⁺ on apoptosis in MDBK cells

Cd²⁺ and Zn²⁺ have both been shown to accelerate apoptosis in dose- and species-dependent manners [16], [19], [20] and we therefore determined the concentrations of these ions that induced toxic effects on MDBK cells. Cells were treated with 10–100 μM of CdCl₂ or 10–200 μM of ZnCl₂ for 12 h and cell survival was analyzed by flow cytometry with staining of annexin V and PI, to detect early and late apoptosis. We observed that 10 μM Cd²⁺ induced mild apoptotic cell damage (Fig. 1A), whereas 50 μM Cd²⁺ caused severe cell death (Fig. 1B). Nearly all cells treated with 100 μM CdCl₂ underwent apoptosis (data not shown). We found that Cd²⁺-triggered cytotoxicity was both time- and dose-dependent. Compared to these cytotoxic effects of Cd²⁺, addition of 10 or 50 μM Zn²⁺ did not induce apoptosis (Fig. 1B). Based on these findings, 10 μM or 50 μM Cd²⁺ was used subsequently to induce mild or severe toxicity, respectively, for investigation of the possible protective role of Zn²⁺ supplementation (10 or 50 μM).

Figure 1. Effects of Zn²⁺ and Cd²⁺ on MDBK apoptosis.

(A) MDBK cells were treated with 10 μM CdCl₂, alone or in combination with ZnCl₂ (0, 10, or 50 μM), for 12 h. Apoptotic cell death was quantified by flow cytometry following double staining with propidium iodide (PI) and a fluorescein isothiocyanate (FITC)-conjugated annexin V antibody. Control cells were treated with medium. (B) MDBK cells were treated with CdCl₂ alone (0, 10, or 50 μM) or in combination with ZnCl₂ (0, 10, or 50 μM) for the indicated time periods. The percentage of PI- or annexin V-positive apoptotic cells was quantified by flow cytometry. The data were expressed as the mean ± SD (n = 4). #P<0.05, ##P<0.01, and ###P<0.001 for the comparison with the medium-treated control group; *P<0.05 and **P<0.01 compared to cells exposed to Cd²⁺ only (10 or 50 μM).

When cells were exposed to 10 μM CdCl₂, supplementation with 10 μM or 50 μM Zn²⁺ produced comparable improvements in cell survival up to 24 h after treatment (Fig. 1B). In contrast, Zn²⁺-mediated protection could only be detected at earlier time points (3 h and 6 h) when 50 μM CdCl₂ was present. After 12-h and 24-h exposure to 50 μM Cd²⁺, addition of 50 μM Zn²⁺ even intensified Cd²⁺-induced cytotoxicity (Fig. 1B). These results suggested that Zn²⁺ prevented mild Cd²⁺-induced cell damage.

Effect of Zn²⁺ on Cd²⁺-induced ROS accumulation in MDBK cells

ROS accumulation plays both direct and indirect roles in inducing apoptosis [21]. We therefore analyzed the levels of ROS, as detected by DCFH-DA conversion to DCF, in MDBK cells exposed to Cd²⁺ and/or Zn²⁺ at the indicated concentrations. As shown in Fig. 2A, strong DCF fluorescence intensity was observed in cells treated with 10 μM Cd²⁺ for 6 h, whereas the addition of Zn²⁺ alone did not cause an obvious increase in ROS levels. Co-treatment with Cd²⁺ and Zn²⁺ led to a lower ROS level in the MDBK cells, as compared to treatment with Cd²⁺ alone.

Figure 2. Effect of Zn²⁺ on Cd²⁺-induced accumulation of reactive oxygen species (ROS) in MDBK cells.

(A) MDBK cells were treated with 10 μM CdCl₂, alone or in combination with ZnCl₂ (0, 10, or 50 μM), for 6 h. Intracellular ROS accumulation was assessed by DCFH-DA staining and imaged using a fluorescence microscope at ×200 magnification. (B) Cells were treated with Zn²⁺ and/or Cd²⁺ as indicated. Intracellular ROS was determined by flow cytometry following staining with DCFH-DA and the percentage of ROS-positive cells was quantified. The data represent the mean ± SD (n = 4). ##P<0.01, and ###P<0.001, compared to the medium-treated control group; *P<0.05 and **P<0.01 compared to the cells exposed to Cd²⁺ alone (10 or 50 μM).

A detailed time course analysis of the protective effects of Zn²⁺ on Cd²⁺-induced ROS accumulation was performed. MDBK cells were exposed to the indicated concentrations of Cd²⁺ and Zn²⁺ for 3–24 h, and the percentage of ROS positive cells was quantified by flow cytometry analysis. As shown in Fig. 2B, ROS accumulation during MDBK exposure to 10 μM Cd²⁺ was time-dependent, and ROS accumulation peaked at 24 h. However, exposure to 50 μM Cd²⁺ caused a sharp rise in the ROS level at 6 h. Prolonged exposure did not result in an increased number of ROS-positive cells, which might reflect the Cd²⁺-induced cell death observed in Fig. 1B. Addition of either 10 μM or 50 μM Zn²⁺ was associated with reduced ROS levels in cells exposed to 10 μM Cd²⁺ at all of the time points tested. When cells were exposed to 50 μM Cd²⁺, the presence of Zn²⁺ reduced ROS accumulation slightly at earlier time points (up to 12 h), but not at 24 h (Fig. 2B). Consistent with the apoptosis data presented in Fig. 1B, the presence of 50 μM Zn²⁺ even synergized Cd²⁺-induced ROS in MDBK cells; this was in agreement with previous observations in other species, where relatively high concentrations of Zn²⁺ led to elevated levels of ROS and cytotoxicity [22].

Effect of Zn²⁺ on Cd²⁺-induced mitochondrial depolarization

Mitochondria are both the source and the target of ROS [21], and we therefore explored the impact of Cd²⁺ exposure on mitochondrial MMP. MDBK cells were exposed to Cd²⁺ and/or Zn²⁺ for 6 h prior to MMP evaluation by JC-1 fluorescence imaging of (Fig. 3A). Exposure to 10 μM or 50 μM Cd²⁺ caused green JC-1 fluorescence in MDBK cells, indicating that the MMP was reduced and that the mitochondria were therefore depolarized. Addition of Zn²⁺ reversed this Cd²⁺-induced decline in MMP, with the best effect observed in the presence of 50 μM of Zn²⁺. Treatment with 10 μM or 50 μM Zn²⁺ alone did not obviously alter MMP in MDBK cells.

Figure 3. Effect of Zn²⁺ on Cd²⁺-triggered mitochondrial depolarization.

(A) MDBK cells were treated with Cd²⁺ alone (0, 10, 50 μM), or in combination with Zn²⁺ (0, 10, 50 μM), for 6 h. Mitochondrial membrane polarization (MMP) was determined by staining with JC-1 probe and imaging using a fluorescence microscope at ×200 magnification. (B) MDBK cells were treated as described above for the indicated time periods. The percentage of green fluorescent cells (indicating reduced MMP) was quantified by flow cytometry. Values shown are the mean ± SD (n = 4). #P<0.05, ##P<0.01, and ###P<0.001 compared to the medium-treated control group; *P<0.05 and **P<0.01 compared to the cells exposed to Cd²⁺ alone (10 or 50 μM).

To examine the time-course of this Zn²⁺-mediated protection of MMP, the amounts of green fluorescence-emitting cells were quantified by flow cytometry over time (3–24 h, Fig. 3B). This showed that Cd²⁺ exposure led to dose- and time-dependent mitochondrial depolarization. At all of the examined time points, exposure to 50 μM Cd²⁺ resulted in around ten-fold more mitochondrial damage than did exposure to 10 μM Cd²⁺. Consistent with our previous observations, the protective effects of Zn²⁺ supplementation on MMP were conditional upon the Cd²⁺ concentration and the exposure time. Zn²⁺ afforded the most obvious protection against the lower Cd²⁺ concentration (10 μM) or at the early stages of exposure to the higher Cd²⁺ concentration (50 μM). Taken together, these results further illustrated that Zn²⁺ had the capacity to counteract Cd²⁺-induced toxicity in MDBK cells. However, exposure to high levels of Cd²⁺ could result in irreversible damage to bovine renal cells, which would not be alleviated by Zn²⁺ administration.

Effect of Zn²⁺ on MTF-1 and MT expression in Cd²⁺-exposed MDBK cells

Intracellular MTs are essential for Cd²⁺ detoxification, and the zinc finger transcription factor MTF-1 plays a critical role in metal-induced MT transcription. We therefore determined the impact of Cd²⁺ and/or Zn²⁺ exposure on MT-1, MT-2, and MTF-1 mRNA levels in MDBK cells using real-time PCR. As shown in Fig. 4A, 6-h exposure to Cd²⁺ or Zn²⁺ alone led to comparable up-regulation of MT-1 and MT-2 mRNA levels. Moreover, incubation with both Zn²⁺ and Cd²⁺ substantially enhanced transcription of these MTs. Co-treatment with both Zn²⁺ and Cd²⁺ significantly enhanced MTF-1 mRNA levels in MDBK cells (Fig. 4A). We also observed increased levels of MT and MTF-1 proteins in MDBK cells following 6-h exposure to Cd²⁺ and Zn²⁺ using fluorescence imaging (Fig. 4B).

Figure 4. Zn²⁺ and Cd²⁺ increased expression of MTs and MTF-1.

MDBK cells were treated with either Cd²⁺ alone (0, 10, 50 μM), or in combination with Zn²⁺ (0, 10, 50 μM), for up to 24 h. (A) MT-1, MT-2, and MTF-1 mRNA levels were determined by real-time PCR after 6-h treatments. Target gene threshold cycle (Ct) values were normalized to those of the reference gene (GAPDH) and the data were expressed as fold change over the medium-treated control sample, which was set as 1. The results are presented as the mean ± SD (n = 4). #P<0.05 compared to the medium-treated control group; *P<0.05, and **P<0.01 compared to the cells exposed to Cd²⁺ alone (10 or 50 μM). (B) Protein levels of MTs and MTF-1 after 6-h treatments were evaluated by fluorescence microscopy at ×400 magnification following staining with anti-MTF-1 or anti-MT antibodies. (C) Protein levels of MTs and MTF-1 in response to Zn²⁺ treatment (10 or 50 μM) at the indicated time points, as determined by western blot analysis. (D) Protein levels of MTs and MTF-1 in response to exposure to both Zn²⁺ and Cd²⁺ (10 and 50 μM, respectively) at the indicated time points, as determined by western blot analysis. In (C) and (D), expression of β-actin was used as the loading control.

To study the kinetics of Zn²⁺- and Cd²⁺-mediated up-regulation of MT-1, MT-2, and MTF-1, MDBK cells were exposed to the same conditions as those presented in Fig. 4A prior to quantification of MTs and MTF-1 protein levels by western immunoblotting at a range of time points. As shown in Fig. 4C, levels of the MTs and MTF-1 proteins peaked dose-independently after 24 h and 12 h Zn²⁺ treatment, respectively. The addition of Zn²⁺ to Cd²⁺-exposed cells enhanced the MTs and MTF-1 protein levels in a dose- and time-dependent manner (Fig. 4D). These findings were consistent with the observations presented in Fig. 4A and indicated that Cd²⁺ and Zn²⁺ stimulated expression of MTs and MTF-1 in a synergistic manner.

Effects of Zn²⁺ administration on Cd²⁺ accumulation in MDBK cells

As Zn²⁺ protected MDBK cells from Cd²⁺-induced cytotoxicity and up-regulated MT proteins, we next explored whether Zn²⁺ administration was able to reduce intracellular Cd²⁺ accumulation. MDBK cells were treated with 10 μM (Fig. 5A) or 50 μM (Fig. 5B) Cd²⁺ in the presence or absence of Zn²⁺ at indicated concentrations (0 to 50 μM), and the intracellular levels of Zn²⁺ and Cd²⁺ were determined over time using ICP-MS. In cells exposed to 10 μM Cd²⁺, addition of Zn²⁺ resulted in a dose-dependent reduction in the intracellular Cd²⁺ level, which was associated with increased levels of intracellular Zn²⁺ (Fig. 5A). In contrast, the inhibitory impact of Zn²⁺ supplementation on Cd²⁺ uptake was not obvious in cells exposed to a high concentration of Cd²⁺ (50 μM). Moreover, exposure to a high concentration of Zn²⁺ (50 μM) even tended to increase intracellular Cd²⁺ levels (Fig. 5B, right panel). Notably, the intracellular Zn²⁺ level under these circumstances was comparable to that of control samples after 24 h treatment, even though transient Zn²⁺ absorption was detected (Fig. 5B, left panel). These results indicated that Zn²⁺ supplementation was able to prevent Cd²⁺ uptake into MDBK cells, but its efficacy was dependent on the level of Cd²⁺ exposure.

Figure 5. Quantification of intracellular Cd²⁺ and Zn²⁺.

MDBK cells were treated with 10 μM (A) or 50 μM (B) Cd²⁺, alone or in combination with Zn²⁺ (0, 10, or 50 μM). At the indicated time points, the intracellular levels of Zn²⁺ and Cd²⁺ (ng/1×10⁴ cells) were determined by ICP-MS, as described in the Materials and Methods section. The results represent the mean ± SD (n = 4). *P<0.05, compared to the cells without Zn²⁺ supplementation.

Discussion

This study demonstrated that Cd²⁺ exposure was cytotoxic to a bovine cell line (MDBK) and indicated that Zn²⁺ supplementation had protective effects against this Cd²⁺-mediated cytotoxicity. In line with previous observations in other species [23], [24], Cd²⁺ triggered MDBK apoptotic cell death in a time- and dose-dependent manner. This was associated with a Cd²⁺-induced increase in ROS accumulation in these cells, and mitochondrial depolarization. Addition of Zn²⁺ counteracted this Cd²⁺-induced oxidative stress and restored the MMP; effects which may explain the Zn²⁺-mediated protection against Cd²⁺-induced apoptosis death in these cells. Our findings also indicated that Zn²⁺ was less protective in cells exposed to the higher Cd²⁺ concentration used in this study (50 μM), especially when cells were treated with prolonged Cd²⁺ exposure (>12 h), indicating that early administration of Zn²⁺ is important in the prevention of Cd²⁺ toxicity.

Mitochondria are attacked by ROS, and ROS elimination may therefore protect mitochondria from structural damage and reduce the release of pro-apoptotic factors. Zn²⁺ plays an essential role in maintaining the wild-type conformation of copper-zinc superoxide dismutase 1 (SOD1), which plays a key role in the antioxidant defense system [25]. Moreover, SOD1 protects cells from apoptosis by inhibiting caspase-9 activation and release of mitochondrial cytochrome c [25], and by reducing chronic endoplasmic reticulum stress [26]. Another endogenous defense system against metal-induced oxidative stress involves glutathione (GSH) and GSH-associated enzymes [27]. GSH prevents Cd²⁺ intake through down-regulating expression of the Cd²⁺ transporter ZIP8 [28]. Effect of Zn on maintaining normal GSH levels has been investigated in several species such as human, mouse, fish and hamster [17], [29]–[31]. It would therefore be interesting to characterize the role of SOD1 and GSH-associated enzymes in Zn²⁺-mediated protection from Cd²⁺ cytotoxicity in future studies.

The conserved function of MTs in Cd²⁺ detoxification has been demonstrated in many species including yeast, zebra fish, mouse, and humans [7], [32]. However, large tissue- and individual-variation in the expression of MTs and their transcription regulatory factor, MTF-1, have been observed and these may affect the cellular response to Cd²⁺-induced toxicity [33]. In this study, we systematically assessed the regulation of MTs and MTF-1 upon exposure to Cd²⁺ and/or Zn²⁺ in MDBK cells. Our results showed that treatment with either Zn²⁺ or Cd²⁺ alone resulted in comparable up-regulation of MTs, indicating that they may be involved in MDBK cellular defense against toxic levels of heavy metals, consistent with previous reports in other species [10]. Notably, co-exposure to both Zn²⁺ and Cd²⁺ resulted in a remarkable dose- and time-dependent increase of MT-1 and MT-2 levels in MDBK cells. In line with previous in vivo and in vitro studies demonstrating that MTF-1 was the major regulator of MT gene expression, we also observed strong up-regulation of MTF-1 in MDBK cells exposed to Zn²⁺ and Cd²⁺. Our results indicated that the presence of Cd²⁺ promoted Zn²⁺-mediated up-regulation of MTF-1 and MTs. Cd²⁺ has a high binding affinity to MTs and may displace MT-bound Zn²⁺, triggering activation and nuclear translocation of MTF-1. Nevertheless, we cannot exclude the role of other factors affecting MT gene expression, such as activation of the antioxidant response element or epigenetic modification of the MT promoters [34].

Although co-exposure to Cd²⁺ and Zn²⁺ led to dose- and time-dependent up-regulation of MT protein levels, this did not protect MDBK cells from intracellular accumulation of Cd²⁺ or from apoptosis during exposure to a high Cd²⁺ concentration (50 μM), suggesting that MT-mediated sequestration may not be sufficient to prevent Cd²⁺ toxicity in these cells. However, we found that when cells were exposed to low levels of Cd²⁺ (10 μM), supplementation of Zn²⁺ could efficiently reduce intracellular Cd²⁺ content, probably through MT-mediated detoxification. Our finding that this was not observed when cells were exposed to high levels of Cd²⁺ (50 μM) was consistent with a previous report in a rat model [35]. Moreover, the intracellular level of Zn²⁺ even decreased to basal levels at 24 h after challenge, indicating that high levels of Cd²⁺ disturbed Zn²⁺ uptake in MDBK cells; this finding was also consistent with a previous report in a rat model [36]. These results also explained our findings that Zn²⁺ supplementation provided notable protection against Cd²⁺-triggered cytotoxicity at early time points or upon exposure to the lower concentration of Cd²⁺ tested (10 μM).

Collectively, the results from this study revealed that Zn²⁺ had protective effects against Cd²⁺-induced cytotoxicity in MDBK cells. We further demonstrated that Zn²⁺ administration efficiently reduced Cd²⁺ uptake in cells exposed to 10 μM Cd²⁺. These findings fill a current gap in our understanding of the role of Zn²⁺ supplementation in maintaining animal health; dietary Zn²⁺ may help to prevent Cd²⁺ accumulation and toxicity in cattle and other farm animals. Further in vivo investigations to evaluate the most effective dosage are warranted.

Supporting Information

Table S1

Real-time polymerase chain reaction primers.

(DOCX)

Funding Statement

This study was supported by the Fundamental Research Funds for the Central Universities, Huazhong Agricultural University Scientific & Technological Self-innovation Foundation, and the Chinese Agricultural Research Systems (CARS-38). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.