Differential regulation of zebrafish metallothionein-II (zMT-II) gene transcription in ZFL and SJD cell lines by metal ions.

Two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5'-flanking region (1514bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) and caudal fin (SJD) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn(2+) and that this Zn(2+) induction was dose-dependent. Further transient gene expression assay of the zMT-IIA gene promoter was carried out to study the effects of various metal ions (Zn(2+), Cd(2+), Cu(2+), Hg(+), As(3+), As(5+), Cr(3+) and Cr(6+)), and Zn(2+) and Cd(2+) were found to be the most efficient MT gene inducers of zMT-II. As(3+) was a weak inducer of zMT-II in the two cell lines, and Hg(+) caused significant induction only in the SJD cells. No significant induction was found in the other metal ion exposures. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn(2+) or Cd(2+) treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein.

Cytotoxicities and induction of metallothionein (MT) and metal regulatory element (MRE)-binding transcription factor-1 (MTF-1) messenger RNA levels in the zebrafish (Danio rerio) ZFL and SJD cell lines after exposure to various metal ions.

Using zebrafish liver (ZFL) and caudal fin (SJD) cell-line models, the induction of metallothionein (MT) and metal regulatory element-binding transcription factor-1 (MTF-1) mRNA levels by various metal ions (Zn(2+)(,) Cd(2+), Cu(2+), Hg(+), As(3+), As(5+), Cr(3+) and Cr(6+)) were studied using the real-time PCR. The LC(50)-24h values of the metal ions were determined for the two cell lines prior to their exposure to different concentrations (10%, 25%, 50%, 75% and 100% LC(50) values) of the heavy metal ions for RNA assay. The two cell lines were sensitive to As(3+), Cd(2+) and Hg(+). Zn(2+)and Cu(2+) were moderately toxic, and As(5+) and Cr(3+) were less toxic to both cell lines. Each of the metal ions tested was found to cause significant induction of the mRNA levels in the SJD cells. Only Zn(2+), Cd(2+), Cu(2+), Hg(2+) and As(3+) caused significant induction of MT mRNA levels in ZFL cells. Zn(2+)and Cd(2+) were efficient MT inducers in the two cell lines, but As(3+) and As(5+) are strong inducers only in the SJD cell line. In both cell lines, Cu(2+) and Hg(2+) are moderate inducers, and Cr(3+) and Cr(6+) were weak inducers of MT mRNA. MTF-1 induction is believed to be insufficient to cause MT gene induction, but As(3+) and Cd(2+) induced MTF-1 in ZFL cell line. Cd(2+) was the most efficient inducer of MT in the cell lines, and also induced MTF-1 levels with clear dose-responsiveness in SJD cell line. These results indicated that MT induction can occur without MTF-1 induction for many metal ions such as Zn(2+) and Cu(2+).

Metallothionein gene expression in zebrafish embryo-larvae and ZFL cell-line exposed to heavy metal ions.

The aim of this study is to investigate the induction of zebrafish metallothionein (zMT) gene expression following the administration of different metal ions using in vivo and in vitro models. The zebrafish embryo-larvae were used for the in vivo study, and MT gene expression was studied during the development from fertilization (8hpf) to embryo-larval stage using real-time PCR. The LC50 values and zMT mRNA levels were also measured in embryo-larvae exposed to various metal ions. The general trend of 24 h LC50 values as determined is Cu2+ < Hg2+ < Cd2+ << Zn2+. However, Hg2+ was found to be the most potent metal inducer with the highest level of zMT mRNA induction (40-50 folds) in 8hpf embryo-larvae, followed by Cd2+ (approximately 20 folds); Cu2+ and Zn2+ only gave approximately 5 fold of induction. In the in vitro study of ZFL cell-line, Cd2+ is the most potent inducer of zMT mRNA (up to 250 folds), Cu2+ and Zn2+ gave similar potency of approximately 50-100 folds, and Hg2+ gave approximately 40-50 folds of zMT mRNA levels over the control group.