Figures
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.
Citation: Zhang D, Liu J, Gao J, Shahzad M, Han Z, Wang Z, et al. (2014) Zinc Supplementation Protects against Cadmium Accumulation and Cytotoxicity in Madin-Darby Bovine Kidney Cells. PLoS ONE 9(8): e103427. https://doi.org/10.1371/journal.pone.0103427
Editor: Rafael Moreno-Sanchez, Instituto Nacional de Cardiologia, Mexico
Received: January 10, 2014; Accepted: July 2, 2014; Published: August 8, 2014
Copyright: © 2014 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: 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.
Competing interests: The authors have declared that no competing interests exist.
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).
(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.
(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.
(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).
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.
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.
https://doi.org/10.1371/journal.pone.0103427.s001
(DOCX)
Author Contributions
Conceived and designed the experiments: J. Li DZ. Performed the experiments: DZ J. Liu. Analyzed the data: DZ J. Liu HS. Contributed reagents/materials/analysis tools: JG MS ZH ZW. Wrote the paper: DZ J. Li HS.
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