Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

Abstract

Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion.

Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes

“…It has been reported that CHELEX causes a weaker form of zinc deficiency than TPEN, which might be reflected by a lesser impact on phagocytosis. 25 Both kinds of zinc deprivation elevate phagocytosis of GFP-S. pneumoniae, even though the differences do not reach a statistical significance. The fluorescence of GFP-S. pneumoniae is lower than that of DSred-E. coli and GFP-S. aureus (data not shown), most likely due to lower expression of GFP.…”

“…Moreover, the findings presented in Table 2 differ from the literature with regard to the removal of several other elements, indicating that different protocols can lead to differential depletion of trace elements from cell culture media. Copper was reported not to be depleted from culture medium by treatment with CHELEX, 25 whereas ICP-MS showed a reduction of copper by roughly 40% in the CHELEX-treated media compared to RPMI control . On the other hand, manganese was previously found to be removed by CHELEX, 25 but its levels were unaffected by CHELEX treatment in the present study.…”

Parameters Influencing Zinc in Experimental Systems in Vivo and in Vitro

“…Common chelators are CHELEX resin, TPEN (N,N,N 1 ,N 1 -tetrakis (2-pyridylmethyl) ethylenediamine), DTPA (diethylenetriamine pentaacetic acid), and also others such as BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N 1 ,N 1 -tetraacetic acid), EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N 1 ,N 1 -tetraacetic acid). Even if some of these chelators show a high selectivity for one metal, e.g., TPEN for zinc, they do not always only bind their target metal, but others as well [22], making it difficult to assign an observed effect to altered zinc conditions. Therefore, a control with the same saturated chelator is essential.…”

Chromium (VI)-induced transformation is enhanced by Zn deficiency in BALB/c 3T3 cells

“…40 No. 3 0.6 μM, respectively (Cho et al, 2007). Whole cell extract from three-day cultures with normal medium, Zndeficient medium, or Zn-deficient medium plus 50 μM Zn were subjected to Western blot analysis with anti-MT and anti-β-actin antibodies.…”