The overall objective of these studies is to employ the zebrafish (Danio rerio) model to understand the relative role of multiple cytochrome P450 1 (CYP1) and aldo-keto reductase (AKR) enzymes in chemically dependent oxidative stress and DNA damage during development. Oxidative stress may be a key mechanistic point at which pathways leading to toxicity converge. Xenobiotic metabolizing enzymes may contribute to formation of reactive oxygen species (ROS) by toxic/carcinogenic halogenated and polynuclear aromatic hydrocarbons that are aryl hydrocarbon receptor agonists. These compounds elicit severe defects during development, and recent studies suggest a role for CYP1A in some of those effects via uncoupling of the CYP catalytic cycle, or through formation of metabolites that undergo redox cycling resulting in ROS generation. The hypothesis to be addressed is that the multiple CYP1 gene family members, including CYP1A, CYP1B and the novel CYP1C genes, as well as AKR, are involved in generation of ROS leading to oxidative damage and further effects during development in zebrafish. We will test this hypothesis by evaluating the sources and consequences of ROS generation. In the Specific Aims we will: 1) Evaluate changes in expression of CYP1s and AKRs and ROS generation, in relation to global changes in gene expression, the oxidative stress response and DNA damage in embryos exposed to tert-butylhydroquinone or the protoxicants (2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene). 2) Establish the temporal and cellular patterns of expression of CYP1 A, CYP1B and novel zebrafish CYP1C and AKR genes and their regulation by chemicals during development. 3) Determine the roles of cloned and heterologously expressed zebrafish CYP and AKR in generation of ROS. 4) Establish that CYP1s and AKR contribute significantly to ROSformation in intact cells. 5) Employ morpholino technology to knock down the expression of the CYP1A, CYP1B, CYP1C and AKR genes in embryos, and determine the effect of gene elimination on toxicant-induced formation of ROS in vivo. Knock-down fish will be examined for a) ROS formation, b) altered gene expression measured with microarrays and by PCR analysis of expression of targeted selected genes including a novel zebrafish retrotransposon potentially regulated by oxidative stress, and c) the occurrence of DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES015912-04
Application #
7774367
Study Section
Special Emphasis Panel (ZES1-SET-A (P1))
Program Officer
Chadwick, Lisa
Project Start
2007-05-10
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
4
Fiscal Year
2010
Total Cost
$625,947
Indirect Cost
Name
Woods Hole Oceanographic Institution
Department
Biology
Type
Other Domestic Higher Education
DUNS #
001766682
City
Woods Hole
State
MA
Country
United States
Zip Code
02543
Wincent, Emma; Stegeman, John J; J├Ânsson, Maria E (2015) Combination effects of AHR agonists and Wnt/?-catenin modulators in zebrafish embryos: Implications for physiological and toxicological AHR functions. Toxicol Appl Pharmacol 284:163-79
Stegeman, John J; Behrendt, Lars; Woodin, Bruce R et al. (2015) Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast. Biochim Biophys Acta 1850:2340-52
Hahn, Mark E; Timme-Laragy, Alicia R; Karchner, Sibel I et al. (2015) Nrf2 and Nrf2-related proteins in development and developmental toxicity: Insights from studies in zebrafish (Danio rerio). Free Radic Biol Med 88:275-289
Kubota, Akira; Goldstone, Jared V; Lemaire, Benjamin et al. (2015) Role of pregnane X receptor and aryl hydrocarbon receptor in transcriptional regulation of pxr, CYP2, and CYP3 genes in developing zebrafish. Toxicol Sci 143:398-407
Tokunaga, Saimi; Stegeman, John J (2014) Elimination of nonspecific bands in non-radioactive electrophoretic mobility shift assays using the digoxigenin system. Anal Biochem 465:70-2
Hahn, Mark E; McArthur, Andrew G; Karchner, Sibel I et al. (2014) The transcriptional response to oxidative stress during vertebrate development: effects of tert-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin. PLoS One 9:e113158
Teraoka, Hiroki; Okuno, Yuki; Nijoukubo, Daisuke et al. (2014) Involvement of COX2-thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish. Aquat Toxicol 154:19-26
Zanette, Juliano; Jenny, Matthew J; Goldstone, Jared V et al. (2013) Identification and expression of multiple CYP1-like and CYP3-like genes in the bivalve mollusk Mytilus edulis. Aquat Toxicol 128-129:101-12
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
Nelson, David R; Goldstone, Jared V; Stegeman, John J (2013) The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s. Philos Trans R Soc Lond B Biol Sci 368:20120474

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