Zinc pre-exposure improves Zn resistance by demethylation of metallothionein 2 and transcription regulation of zinc-regulatory genes in zebrafish ZF4 cells

Abstract

Mild zinc (Zn) pre-exposure can promote Zn resistance of organism, but the underlying molecular mechanisms are largely unknown. Two experiments were performed using zebrafish ZF4 cells, including short-term and long-term Zn pre-exposure experiments. In the short-term test, the cells were pre-exposed to 100 µM Zn for 24 h, transferred into fresh medium with 4.4 µM Zn for 24 h, and then re-exposed to 250 µM Zn. In the long-term test, the cells were pre-exposed to 100 µM Zn intermittently for 10 passages (3 days per passage), transferred into fresh medium with 4.4 µM Zn for 5 passages, and then re-exposed to 250 µM Zn. Both pretreatments resulted in higher resistance to 250 µM Zn. Exposure to 250 µM Zn caused a more than 2-fold increase in Zn content without Zn pretreatment but did not affect Zn content in the Zn pretreated cells. The Zn pretreated cells had low methylation levels of the metal-response element (MRE) at locus -87 in the promoter of mt2 (metallothionein 2). The up-regulated mRNA expression of Zn-regulatory genes (mtf-1, mt2, slc30a1a, slc30a4, slc30a5, slc30a6 and slc30a7) in the long-term Zn pretreated cells and mt2, slc30a4, slc30a6 and slc30a7 in the short-term Zn pretreated cells were observed. Exposure to 250 µM Zn in combination with the Zn pretreatments up-regulated mRNA expression of these genes and reduced methylation levels of the MRE compared with 250 µM Zn alone and the control. Taken together, the data suggested that demethylation of MRE in the promoter of mt2 and transcriptional induction of mt2 and Zn exporter genes offered Zn resistance in fish ZF4 cells. The traditional toxicological evaluation based on continuous exposure may overestimate the risk of fluctuating concentrations of Zn in the environment.