Limits...
Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

Zhang D, Liu J, Gao J, Shahzad M, Han Z, Wang Z, Li J, Sjölinder H - PLoS ONE (2014)

Bottom Line: 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+.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, PR China.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126686&req=5

pone-0103427-g004: 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.

Mentions: 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).


Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

Zhang D, Liu J, Gao J, Shahzad M, Han Z, Wang Z, Li J, Sjölinder H - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126686&req=5

pone-0103427-g004: 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.
Mentions: 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).

Bottom Line: 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+.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, PR China.

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.

Show MeSH
Related in: MedlinePlus