The long-range goal of this research project is to develop a biological monitor of aquatic environmental pollution. Specifically, we propose to produce lines of transgenic fish in which DNA response elements that respond to a wide variety of environmental pollutants are able to activate an easily assayable reporter gene. We have chosen the response elements of the CYP1A1 (cytochrome P1450), NM01 (NAD(P)H:menadione oxidoreductase; DT diaphorase; quinone reductase; azo dye reductase) and MT (metallothionein) genes. In the mammal, the CYP1A1 gene is known to be controlled by aromatic hydrocarbon response elements (AhREs) that respond to a wide variety of polycyclic hydrocarbons and halegenated planar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. The NM01 gene is known to respond to quinones and a wide variety of other oxidants (potent electrophiles) via an electrophile response element (EpRE). The mammalian (as well as trout) MT gene is known to be regulated by a metal response element (MRE) and respond to heavy metals such as mercury, copper, nickel, cadmium and zinc. For the 3 years of this grant application, we propose to: (1) determine the ability of AhRE, EpRE or MRE sequences from well- characterized mouse and trout polycyclic hydrocarbon-, oxidant-, and metal-responsive gene regulatory regions to modulate transcription of the luciferase (luc) gene in zebrafish cell cultures in response to these pollutants; and (2) generate transgenic zebrafish in which the luc gene is expressed, in order to define the limits of luminescence detection in living adult albino zebrafish. The final product of this project will be a sentinel for biological monitoring of environmental pollution capable of differentiating chemical classes within a complex contaminant mixture with an easily assayable reporter gene. This will provide an alternative model which uses a lower vertebrate for the accurate assessment of environmental hazards to human health.
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