Oxidative stress resulting from environmental exposures is associated with a variety of human diseases ranging from chemical teratogenesis to cardiovascular and neurodegenerative diseases. Developing animals appear to be especially sensitive to chemicals causing oxidative stress. The expression and inducibility of anti- oxidant defenses are critical factors affecting susceptibility to oxidants at these early life stages, but the ontogenic development of these responses in embryos is not well understood. In adult animals, oxidants initiate an anti-oxidant response by activating NF-E2-related factor 2 (NRF2) and related proteins, which bind to the anti-oxidant response element and activate transcription of genes such as glutathione S-transferases, NAD(P)H-quinone oxidoreductase, glutamyl-cysteine ligase, and superoxide dismutase. The overall objective of the research proposed here is to elucidate the mechanisms by which vertebrate embryos respond to oxidative stress during development. We will test the central hypothesis that responsiveness to oxidative stress and the set of regulated genes vary during development. Because of these developmental differences, some stages may be more sensitive to oxidative stress-induced damage. These studies will be performed in vivo using embryos of the zebrafish (Danio rerio), a valuable model in which to examine mechanisms of toxicity in developing animals and to screen chemicals for developmental toxicity.
Aim 1 will use transcriptional profiling and phenotypic anchoring to identify the core set of genes that comprise the oxidative stress response in embryos, establish how the responsiveness and composition of the core set of oxidant-responsive genes vary with developmental stage of embryos, and determine how the timing and gene profile of the oxidative stress response differ in embryos exposed to structurally and mechanistically distinct activators of NRF2 (tBHQ, diquat, sulforaphane).
Aim 2 will elucidate the roles of different NRF paralogs in the transcriptional response to oxidative stress during development in embryos in vivo, using targeted knock-down of NRF protein synthesis with morpholino oligonucleotides.
Aim 3 will establish the mechanism of regulation of anti-oxidant response genes during embryonic development through computational and in vivo experimental analysis of oxidant-responsive gene promoters, leading to the generation of a stable line of transgenic zebrafish expressing a reporter gene (GFP) in response to oxidative stress. Finally, we will test a set of mammalian developmental toxicants for the ability to activate the transgene in embryos. The results of these studies will establish the composition and ontogeny of the transcriptional response to oxidative stress in vertebrate embryos, elucidate fundamental mechanisms underlying this response, generate tools for screening chemicals for activity as developmental toxicants or antioxidants, and provide insight into the role of oxidative stress in human disease.

Public Health Relevance

) Oxidative stress is involved in a variety of environmentally influenced human diseases ranging from chemical teratogenesis to cardiovascular and neurodegenerative diseases. The proposed research will enhance our basic understanding of how some chemicals interfere with embryonic development by generating oxidative stress and how embryos can mitigate these effects. This research will i) determine the basal expression and inducibility of anti-oxidant defenses during embryonic development, ii) elucidate fundamental mechanisms of the response of vertebrate embryos to oxidative stress, iii) establish a model system for screening chemicals for activity as developmental toxicants or antioxidants, and iv) provide a mechanistic foundation that will facilitate the extrapolation of results obtained in zebrafish to humans, supporting risk assessment and providing insight into the role of oxidative stress in human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES016366-04
Application #
8244524
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Chadwick, Lisa
Project Start
2009-06-15
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
4
Fiscal Year
2012
Total Cost
$370,788
Indirect Cost
$138,951
Name
Woods Hole Oceanographic Institution
Department
Type
DUNS #
001766682
City
Woods Hole
State
MA
Country
United States
Zip Code
02543
Williams, Larissa M; Timme-Laragy, Alicia R; Goldstone, Jared V et al. (2013) Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the zebrafish, Danio rerio. PLoS One 8:e79574
Harbeitner, Rachel C; Hahn, Mark E; Timme-Laragy, Alicia R (2013) Differential sensitivity to pro-oxidant exposure in two populations of killifish (Fundulus heteroclitus). Ecotoxicology 22:387-401
Timme-Laragy, Alicia R; Goldstone, Jared V; Imhoff, Barry R et al. (2013) Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo. Free Radic Biol Med 65:89-101
Timme-Laragy, Alicia R; Karchner, Sibel I; Franks, Diana G et al. (2012) Nrf2b, novel zebrafish paralog of oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2). J Biol Chem 287:4609-27
Hahn, Mark E (2011) Mechanistic research in aquatic toxicology: perspectives and future directions. Aquat Toxicol 105:67-71