Detection of mercury in aquatic environments using EPRE reporter zebrafish

Abstract

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase-green fluorescent protein (LUC-GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl(2) concentrations. GFP expression from the EPRE-LUC-GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl(2). We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl(2) concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.

Fig. 1

Oxidative stress responsive reporter gene. a Under homeostatic conditions, Nrf2 is retained in the cytoplasm and prevented from binding to the EPRE and activating the luciferase–GFP fusion transgene expression. b Under conditions of oxidative stress, Nrf2 is released and translocated into the nucleus where it is able to bind to the EPRE and activate the luciferase–GFP fusion gene, resulting in luciferase (LUC) activity and GFP expression

Fig. 2

Twenty-four exposure at ≥0.3 μM HgCl₂ leads to significant increases in deformities and death in zebrafish embryos. Embryos were treated in groups of ten at 24 hpf for 24 h, then observed for death/deformities. Results are the average values from seven groups for each treatment

Fig. 3

Embryos injected with the EPRE–luciferase–GFP reporter plasmid expressed GFP after 24 h exposure to HgCl₂. Control embryos that were uninjected or injected but untreated did not express GFP (a). HgCl₂-induced GFP expression was most commonly observed in skin cells (b, e), although at treatment, ≥0.3 μM expression was also observed in other cell types including neurons (c, f), muscle cells (c, g), and notochord cells (d, h). Scale bar applies to a–d=200 μm

Fig. 4

Characterization of transgene expression in injected (F0) embryos. a Embryos that survived 24 h HgCl₂ treatment were characterized for the presence or absence of GFP-expressing cells. A greater percentage of embryos expressed GFP at higher concentrations of HgCl₂. b Embryos expressing GFP were scored based on the number of cells expressing GFP as + (one to ten cells expressing), ++ (ten to 100 cells expressing), and +++ (more than 100 cells expressing). A greater number of GFP+ cells/embryos were observed at higher concentrations of HgCl₂. c Embryos expressing GFP were scored for expression in the head, trunk, and/or tail. GFP expression is more widely distributed at higher concentrations of HgCl₂. d Injected embryos treated at 0.3 uM were divided into groups of two based on number of cells expressing GFP as in b. Luciferase activity of these groups was measured. High levels of luciferase activity correlate with high levels of GFP expression. Error bars represent standard error of the mean

Fig. 5

Characterization of transgene induction in stable transgenic lines. a Injected F0 fish have mosaic expression of the transgene. F0 founders transmit the transgene through the germ line to a small fraction of F1 progeny. b Crosses of transgene positive F1 fish results in 3/4 of the progeny carrying at least 1 copy of the transgene. c Luciferase activity was detectable in induced stable line embryos, but at lower levels when compared to injected embryos, although levels were still within the linear range of the assay. There was no visible GFP expression in stable line induced embryos. Transgene expression is diminished at concentrations of HgCl₂ that are correlated with increased mortality. Results are the average values from 3 groups of 8 for each treatment. Error bars represent standard error of the mean