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 microM vs 0.61 +/- 10.15 microM) and Mn (p<0.05) (0.74 +/- 0.01 microM vs 0.12 +/- 0.04 microM). 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 microM) except for in the addition of higher 15 microM ZnCl(2) 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.

Keywords: DTPA; MC3T3-E1 cells; TPEN; Zn depletion; chelexing; metal ion chelators.

Fig. 1

Trace mineral (Zn, Mn, Fe and Cu) composition of various cell culture media components using chelexed FBS in different media (n=2) Different letter superscripts mean significantly different between media composition at p<0.05 by Tukey, one-way ANOVA. [A, αMEM (fresh); B, FBS (fresh); C, chelexed FBS; D, αMEM +10% FBS; E, αMEM +10% chelexed FBS; F, αMEM +10% chelexed FBS +15 µM ZnCl2; G, DMEM; H, DMEM +10% FBS].

Fig. 2

Trace mineral (Zn, Mn, Fe and Cu) composition of osteoblastic cell culture media with various Zn levels using chelexed FBS (n=2) Zn-treated Media (0-15 µM Zn) were prepared as the addition of 0 to 15 µM Zn as ZnCl2 to a-MEM plus chelexed FBS. Different letter superscripts mean significantly different between Zn media treatments at p<0.05 by Tukey, one-way ANOVA.

Fig. 3

Zn and Fe level of osteoblastic cell culture media components using TPEN and DTPA as Zn chelators (n=2) Five µM TPEN and 1.2 mM DTPA were the optimized concentration used to chelate Zn. Different letter superscripts mean significantly different between media composition at p<0.05 by one-way ANOVA. [A, αMEM; B, growth media; C, osteogenic differentiation media + TPEN or DTPA^*; D, 0 µM ZnCl₂ + TPEN or DTPA; E, 3 µM ZnCl₂ + TPEN or DTPA; F, 6 µM ZnCl₂ + TPEN or DTPA; G, 9 µM ZnCl₂ + TPEN or DTPA; H, 12 µM ZnCl₂ + TPEN or DTPA; I, 15 µM ZnCl₂ + TPEN or DTPA].

Fig. 4

Bone-related gene expressions in osteoblastic MC3T3-E1 cells at 5 days of Zn treatment using A) chelexed FBS and B) TPEN in Zn-treated osteogenic differentiation medium Asterisks (^*) mean significantly different between Zn treatments (Zn- vs Zn+) at p<0.05 by one-way ANOVA.