Use of reporter genes and vertebrate DNA motifs in transgenic zebrafish as sentinels for assessing aquatic pollution. (Correspondence).

In a recent paper in EHP, Mattingly and co-workers (1) showed that dioxin-treated transgenic zebrafish [having 1,905 bp of 5' flanking region of the human CYP1A1 gene, driving the jelly fish green fluorescent protein (GFP) reporter gene] displayed inducible GFP in the eye, nose, and vertebra of embryos 48 and 72 hr after fertilization. They first confirmed in zebrafish liver cells that zebrafish transcription factor proteins [e.g. the aromatic hydrocarbon receptor (AHR) and its heterodimeric binding partner the AHR nuclear transporter (ARNT)] must be capable of recognizing and binding to human aromatic hydrocarbon response elements (AHREs) present in the CYP1A1 5' flanking region.

We wish to point out that we have done similar experiments, although they were not cited by Mattingly et al. (1). We first made plasmid constructs using mammalian or trout response elements to drive the firefly luciferase (LUC) reporter gene and showed that transient transfection of the zebrafish ZEM2S cell line with these reporter constructs imparts dose-dependent gene induction upon exposure to a variety of chemicals (2,3). Using the golden mutant zebrafish, which has a decrease in interfering pigmentation, we then developed transgenic fish in which vertebrate DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed; details of our successes and failures in trying to generate stably transformed transgenic zebrafish cell lines have been reported (4). The expression of transgenes in zebrafish in our hands has been quite difficult to maintain past the [F.sub.1] generation, although some laboratories have been more successful (5). However, the short generation time (12 weeks), long life span (2-3 years), and relatively small diploid genome make the zebrafish a very attractive experimental model system.

We began with three DNA motifs that recognize three important classes of foreign chemicals (2). AHREs respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. We first established that zebrafish transcription factors are able to recognize mammalian or trout AHRE, EPRE, or MRE sequences in a dose-dependent and chemical-class--specific manner, and that expression of both the GFP and LUC reporter genes are easily detected in zebrafish cell cultures (2) and in intact live zebrafish (4). As anticipated, some agents gave a response to only one of the three classes, whereas others gave a mixed (AHRE- plus EPRE-mediated or MRE- plus EPRE-mediated) response. We are extending these studies to include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the possible effects of retinoids in the environment.

A very important aspect of this assay is its sensitivity due to the property of bioconcentration that is exhibited to varying degrees in all fish species. Each environmental pollutant is known to be bioconcentrated; for example, [10.sup.-17] M TCDD in a body of water is concentrated 100,000 times (6) to approximately [10.sup.-12] M TCDD in a fish, where it would act upon the AHRE motif and turn on the GFP or LUC reporter gene. Variations in sensitivity of this model system can also be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core consensus response element and flanking regions. This transgenic technology should allow for a simple, exquisitely sensitive, and inexpensive assay for monitoring aquatic pollution.

Daniel W. Nebert
University of Cincinnati Medical Center
Cincinnati, Ohio
E-mail [e-mail]

Gary W. Stuart
Indiana State University
Terre Haute, Indiana

Willy A. Solis
SUGEN, Inc.
South San Francisco, California

Michael J. Carvan, III
Great Lakes WATER Institute
University of Wisconsin-Milwaukee
Milwaukee, Wisconsin

REFERENCES AND NOTES

(1.) Mattingly CJ, McLachlan JA, Toscano WA Jr. Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AHR) function in developing zebrafish (Danio rerio). Environ Health Perspect 109:845-849 (2001).

(2.) Carvan MJ III, Solis WA, Gedamu L, Nebert DW. Activation of transcription factors in zebrafish cell cultures by environmental pollutants. Arch Biochem Biophys 376:320-327 (2000).

(3.) Dalton TP, Solis WA, Nebert DW, Carvan MJ III. Characterization of the MTF-1 transcription factor from zebrafish and trout cells. J Compar Physiol Biochem Part B 126:325-335 (2000).

(4.) Carvan MJ III, Dalton TP, Stuart GW, Nebert DW. Transgenic zebrafish as sentinels for aquatic pollution. Ann NY Acad Sci 919:133-147 (2000).

(5.) Gibbs PD, Schmale MC. GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish. Mar Biotechnol 2:107-125 (2000).

(6.) Frakes RA, Zeeman CQ, Mower B. Bioaccumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by fish downstream of pulp and paper mills in Maine. Ecotoxicol Environ Saf 25:244-252 (1993).