Activation of transcription factors in zebrafish cell cultures by environmental pollutants.
Many classes of environmental pollutants are found at significant levels in the aquatic environment. We are designing a fish model as an inexpensive and efficient system for the assessment of aquatic pollution. Three classes of environmental pollutants-halogenated and nonhalogenated aromatic hydrocarbons, heavy metals, and potent electrophiles-are known to upregulate particular mammalian genes via the activation of specific DNA motifs called aromatic hydrocarbon (AHREs), heavy metal (MREs), and electrophile (EPREs) response elements, respectively. We have made plasmid constructs, using these mammalian or trout response elements to drive the luciferase reporter gene. Here we show 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 within each of these three classes of inducers: [a] (AHRE-mediated) 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, 3,4,5,3',4',5'-hexabromobiphenyl, Aroclor 1254, and benzo[a]pyrene; [b] (MRE-mediated) Cd(2+), Zn(2+), Hg(2+), and Al(3+); and [c] (EPRE-mediated) tert-butylhydroquinone, Hg(2+), Pb(2+), As(3+), Cu(2+), and Cd(2+). As expected, 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. In response to several environmental agents, we found that differences in the electrophoretic mobility shift assay, using the AHRE or MRE as probe, were consistent with the degree of transcriptional activation seen with the reporter constructs. Our data suggest that these reporter constructs might be valuable for the generation of transgenic zebrafish in order to carry out mechanistic and developmental studies of transcriptional activation by environmental contaminants; moreover, such transgenic zebrafish lines might be useful as a sentinel for assessing aquatic pollution.
Carvan
M J
MJ
Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056, USA. carvanmj@uwm.edu
Solis
W A
WA
Gedamu
L
L
Nebert
D W
DW
D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D015342:DNA Probes; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D004742:Enhancer Elements, Genetic / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006219:Halogens / Q000378:metabolism; D006841:Hydrocarbons, Aromatic / Q000378:metabolism / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology; D008024:Ligands; D019216:Metals, Heavy / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects*; D014873:Water Pollutants / Q000494:pharmacology*; D015027:Zebrafish
D015342:DNA Probes; D006219:Halogens; D006841:Hydrocarbons, Aromatic; D006873:Hydroquinones; D008024:Ligands; D019216:Metals, Heavy; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D014873:Water Pollutants; D006861:Hydrogen Peroxide; C018855:2-tert-butylhydroquinone