Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro

Abstract

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as--log(10) [free Zn(2+)]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Figure 1

Free zinc in artificial cerebrospinal fluid (ACSF) and Dulbecco’s modified Eagle’s medium (DMEM) media measured by phase and modulation fluorescence lifetimes of Newport Green as a function of total zinc added. Reproduced with permission from Bozym et al.

Figure 2

Survival (open circles) of adult rat cardiomyocytes and pZn (filled squares) in M199 media as a function of total zinc added

Figure 3

Bright field (panels a, c, e, g, i) and Oregon Green-Annexin V fluorescence (panels b, d, f, h, j) micrographs of PC-12 cells exposed to Neurobasal + B-27 media with 3 μmol/L total, 20 pM free (a, b); 2.6 μmol/L total, 0.5 nmol/L free (c, d); 2.6 μmol/L total, 5 nmol/L free (e, f); 30 μmol/L total, 68 nmol/L free (g, h); and ~1 mmol/L total, 4 μmol/L free (i, j) zinc ions. Fluorescence micrograph exposure times were 30 ms (panel b), 100 ms (d), 500 ms (e), 500 ms (h) and 35 ms (j). (A color version of this figure is available in the online journal)

Figure 4

Time to neuronal quiescence for mouse cortical neurons (open circles) and pZn (filled squares) measured as a function of total added zinc in Dulbecco’s modified Eagle’s medium (DMEM) media plus serum

Figure 5

Survival of HeLa cells in Optimemmedia (open circles) and measured pZn (filled squares) as a function of total zinc added

Figure 6

Twenty-four-hour survival of HT-29 cells in Dulbecco’s modified Eagle’s medium (DMEM) (open circles) and pZn (filled squares) as a function of added zinc ions