Elsevier

Aquatic Toxicology

Volume 89, Issue 2, 29 August 2008, Pages 103-112
Aquatic Toxicology

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

https://doi.org/10.1016/j.aquatox.2008.06.006Get rights and content

Abstract

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 (Zn2+, Cd2+, Cu2+, Hg+, As3+, As5+, Cr3+ and Cr6+) were studied using the real-time PCR. The LC50-24 h 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% LC50 values) of the heavy metal ions for RNA assay. The two cell lines were sensitive to As3+, Cd2+ and Hg+. Zn2+and Cu2+ were moderately toxic, and As5+ and Cr3+ 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 Zn2+, Cd2+, Cu2+, Hg2+ and As3+ caused significant induction of MT mRNA levels in ZFL cells. Zn2+and Cd2+ were efficient MT inducers in the two cell lines, but As3+ and As5+ are strong inducers only in the SJD cell line. In both cell lines, Cu2+ and Hg2+ are moderate inducers, and Cr3+ and Cr6+ were weak inducers of MT mRNA. MTF-1 induction is believed to be insufficient to cause MT gene induction, but As3+ and Cd2+ induced MTF-1 in ZFL cell line. Cd2+ 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 Zn2+ and Cu2+.

Introduction

Metallothionein (MT) is a low molecular weight cysteine-rich intracellular metal-binding protein for the homeostasis of essential metals and detoxification of non-essential metal ions (Andrews, 1990, Andrews, 2000, Kägi, 1991, Palmiter, 1998). MT gene expression is inducible by heavy metal ions and has been shown to play an important role in the sequestration and detoxification of various heavy metal ions (Hogstrand and Haux, 1990, Chan, 1994, Chan, 1995, Adams and Freedman, 2000, Tom and Auslander, 2005, Amiard et al., 2006, Thirumoorthy et al., 2007). However, the induction of MT mRNA by various metal ions remains to be tested for each fish species (Chan et al., 1989, Chan et al., 2006, Eller-Jessen and Crivello, 1998, Cheung et al., 2004, Cheung et al., 2005, Tom and Auslander, 2005; Chan and Chan, 2008).

The molecular mechanism of the metal induction of MT gene expression can be influenced by cis-acting elements in all MT genes, which comprises metal response elements (MREs) (Searle et al., 1984, Searle et al., 1987, Stuart et al., 1984, Stuart et al., 1985, Olsson et al., 1995, Olsson et al., 1998). The metal response element (MRE) is a cis-acting element in target genes for the binding of a trans-acting factor, called MRE-binding transcription factor-1 (MTF-1) (Westin and Schaffner, 1988, Andrews, 2001), which mediates the induction of the MT gene by various metal ions. However, the molecular mechanism of how various metal ions induce MT genes remains to be investigated (Palmiter, 1994, Bittel et al., 1998, Bourdineaud et al., 2006, Marr et al., 2006).

MTF-1 is a ubiquitously expressed zinc finger protein of around 80 kDa essential for basal and metal-induced MT gene transcription (Heuchel et al., 1994, Samson and Gedamu, 1995, Bittel et al., 2000, Lichtlen and Schaffner, 2001, Wang et al., 2004, Li et al., 2006). It was identified later in a wide range of species including mice, humans, mice, chickens, fruit flies (Drosophila melanogaster), pufferfish (Takifugu rubripes), zebrafish (Danio rerio), trout (Oncorhynchus mykiss) and tilapia (Oreochromis spp.) (Radtke et al., 1993, Brugnera et al., 1994, Otsuka et al., 1994, Auf der Maur et al., 1999, Auf der Maur et al., 2000, Carvan et al., 2000, Dalton et al., 2000, Zhang et al., 2001, Chen et al., 2002, Cheung, 2003). Similar to an MRE, MTF-1 is conserved in evolution: human, mouse and fish MTF-1s are highly similar, with a 93% amino acid identity (Brugnera et al., 1994, Lichtlen and Schaffner, 2001) in their zinc finger DNA-binding domains (Brugnera et al., 1994, Cheung, 2003).

Unlike other zinc finger transcription factors, which appear to be constitutively active to bind DNA under normal physiological conditions, MTF-1 is reversibly bound to its target DNA, and the binding is dependent on changes of the availability of free Zn2+ in the cytoplasm (Westin and Schaffner, 1988, Radtke et al., 1993, Dalton et al., 1997, Koizumi et al., 1999, Bittel et al., 2000, Laity and Andrews, 2007). That is, the binding of MTF-1 to the MREs requires Zn2+ activation, and the Zn2+ can enter the nuclei from a dietary source to activate MT gene expression (Cousins and Lee-Ambrose, 1992, Laity and Andrews, 2007). Once the MTF-1 senses free Zn2+, it adopts a reversible DNA-binding conformation. This allosteric change causes exposure of zinc fingers, and the MTF-1 can move to the nucleus and activate gene expression by associating with the gene promoters carrying MREs (Andrews, 2001, Lichtlen and Schaffner, 2001, Jiang et al., 2003). Recent studies have also confirmed that the mediation of MT gene expression starts by the translocation of MTF-1 protein from the cytoplasm to the nucleus and bind with MREs in target genes (Smirnova et al., 2000, Chen et al., 2007). However, whether metal ions can induce the production of MTF-1 in the cytoplasm and how other metal ions induce MT via MTF-1 remains to be elucidated.

The induction of the MT gene by other heavy metal ions, such as arsenic and chromium, has not been investigated. For example, one study found that Cr(VI) could depress the induced human MT-IIA gene by interfering with MTF-1 (Majumder et al., 2003). In fact, MTF-1 serves only as a zinc sensor (Palmiter, 1994) and mediator of hypoxia to induce MT genes (Murphy et al., 1999), and not as a sensor of Cd2+ or oxidative stress (Daniels et al., 2002). However, it is also possible that Zn2+ can function to release MTF-1 from another inhibitor (Palmiter, 1994), and it was demonstrated that MTF-1 bound to other “mediators” to recruit TFIID for activation of MT gene transcription (Marr et al., 2006).

Zebrafish are an ideal in vivo platform to study the relationship between MT gene expression and metal ions. Preliminary studies report that the zebrafish embryo/larva is a sensitive model for exposure experiments and accumulates metal ions (Li et al., 2004, Chen et al., 2004, Chan et al., 2006). A zebrafish MT gene (zMT-II) and its promoter have been previously characterized (Yan and Chan, 2002, Yan and Chan, 2004). Activation of the zMT-II promoter was observed after transfected into SJD cells (zebrafish caudal fin fibroblast cells) (Yan and Chan, 2002), and HepG2 cells (human hepatic cell line) (Yan and Chan, 2004) after exposure to different metal ions. Induction of zMT-II promoter after exposure to Zn2+, Cd2+, Cu2+ and Hg2+ was confirmed, with decreasing potency, whereas hydrogen peroxide, Ni2+, Pb2+ and Co2+ did not cause a significant induction of the zMT gene promoter in either the HepG2 or SJD cells (Yan and Chan, 2002, Yan and Chan, 2004). The goal of this study was to evaluate the effects of 8 metals on the cytotoxicity and expression of MT and MTF-1 in two zebrafish cell lines. Specifically this study focused on the impact of MTF-1 expression and its relation to MT expression and toxicity.

Section snippets

Cell culture

The ZFL cells are zebrafish liver cells obtained from the American Type Culture Collection (ATCC CRL-2643). They were cultured at 28 °C in an incubator with 50% Leibovitz's L-15 medium containing 2 mM l-glutamine (Vitacell 30-2008), 35% Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose and 4 mM l-glutamine (Gibco 12100), 15% Ham's F12 with 1 mM l-glutamine (Gibco 21700), 15 mM HEPES, 0.01 mg/ml insulin (Sigma I-1882), 50 ng/ml epidermal growth factor (EGF), 0.15 g/l sodium bicarbonate and

Toxicities of various heavy metal ions

The LC50 values measured at 24 h for Zn2+, Cd2+, Cu2+, Hg2+, As3+, As5+, Cr3+and Cr6+ for the two cell lines were determined by alarmar blue™ assay. The values of Zn2+, Cd2+, Cu2+ and Hg2+ for the SJD cells were obtained from Yan and Chan (2004) also using alarmar blue™ assay; all other values were determined by the present study. Table 2 shows the cytotoxicities determined for SJD and ZFL cell lines.

The SJD cells were sensitive to Cd2+ and Hg2+, and As3+ and Cr6+ had significant toxic effects

Comparison of metal toxicities in the two cell lines studied

Zn2+ and Cd2+ are efficient inducers of MT, and their mechanistic action on MT gene expression has been studied extensively. However, the role of other heavy metal ions such as Cu2+, Hg2+, As3+, As5+, Cr3+ and Cr6+ in MT gene expression has received little attention, even though these metal ions also pose threat to the general public health and ecosystems (Cherian and Clarkson, 1976, Borgono et al., 1977, Chen et al., 1992, Bertin and Averbeck, 2006, Gonzalez et al., 2006, Uetani et al., 2006).

Acknowledgements

Special thanks to financial support from a Directed Research Grant (MD2041180) entitled “Regulation of metallothionein gene expression in zebrafish” (2006–2007) awarded to KMC by the Medicine Panel, Research Committee, The Chinese University of Hong Kong.

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