The long-term goal of this research is to develop sensitive and reliable in vivo methods for: 1) detecting biologically relevant levels of polycyclic aromatic hydrocarbons (PAHs) in the environment and 2) determining the effects of chronic, sublethal PAH exposure on physiological, biochemical, and molecular function. This second goal is particularly important because world-wide industrial activity is subjecting most organisms to chronic, sublethal levels of pollutants and toxicants. In order to establish minimally acceptable levels of environmental pollution, it is imperative that we understand the biological effects of these chemicals at sublethal, environmentally relevant levels. Therefore, we propose to develop transgenic medaka as a non-mammalian model system for environmental toxicology research utilizing the well characterized hydrocarbon-inducible P450 (CYPIA1) system as the basis for detecting PAH exposure. This approach will provide significant advantages over present techniques for assaying CYPIA1 expression. Once established, we will be able to quantify and characterize CYPIA1 expression in vivo over time by measuring light production. In contrast to presently used techniques, we will not have to sacrifice animals, isolate protein or RNA, or do enzyme assays, immunoblots, or northern blots. Development of a transgenic stock will also produce a homogenous population of fish with known xenobiotic exposure histories. We will then examine the effects of sublethal PAH exposure on reproductive physiology by examining expression of vitellogenin (vg) in response to estradiol treatment. Vitellogenin is the egg-yolk precursor protein and exposure to aromatic hydrocarbons results in reduced level of VG expression, thus jeopardizing reproductive success. The specific objectives of this proposal are: 1) the production of two transgenes, one possession the trout CYPIA1 promoter linked to the green fluorescent protein (GFP)reporter gene and the other possessing the trout VG promoter linked to the bacterial luciferase (LuxAB) reporter gene and, 2) production of transformed cell line and production of transgenic fish possessing these transgenes. Two approaches will be used in the development of these transgenes. The first will be transformation with a dicistronic vector possessing both transgenes. The second approach will be co-transformation with individual vectors. These vectors will initially be tested and confirmed in a fish cell line. Transgenic medaka will be produced by electroporation and a transgenic brood stock will be established. Studies with model PAHs will then be undertaken to examine the effect of sublethal exposure on vitellogenin expression and subsequent reproductive success.
| Rabergh, C M; Vrolijk, N H; Lipsky, M M et al. (2000) Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss). Toxicol Appl Pharmacol 165:195-205 |
| Lu, J K; Burns, J C; Chen, T T (1997) Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes). Mol Mar Biol Biotechnol 6:289-95 |
