The embryonic development of vertebrates is highly susceptible to disruption by exposure to chemicals, such as pharmaceuticals, pesticides, and nanoparticles. Xenobiotics can also cause developmental cardiotoxicity through generation of reactive oxygen species (ROS) and oxidative stress (1), defined as """"""""a disruption of redox signaling and control"""""""" (2). The zebrafish is a powerful model in developmental toxicology and has great potential for use in screening and understanding possible human teratogens, including those that act via oxidative stress. Much remains to be learned about the oxidative stress response during development. We have found that while embryos grow increasingly resistant to pro-oxidant exposure from 18-52 hours post fertilization (hpf), this is followed by a surprising window of heightened sensitivity between 72-96 hpf. Taking a collaborative and multidisciplinary approach, the objective of this proposal is to understand the mechanisms involved in the differential sensitivity to pro-oxidants during embryogenesis.
Aim 1 will identify critical windows of sensitivity, the role of the antioxidant response element transcription factor NRF2, and the impact of pro-oxidant exposure on cardiac progenitor cell fate decisions. These experiments will involve ROS detection by chemiluminescence, cardiac function and cell fate analysis using calcium imaging, as well as embryo survival studies and assessment of the role of NRF2 through loss and gain of function experiments.
Aim 2 will examine the role of glutathione, the most abundant component of antioxidant defenses, using quantitative real-time PCR, measurements of total glutathione, GSH:GSSG ratios, and modulation of GSH content.
Aim 3 will examine the contribution of a physiologic increase in respiration and associated ROS towards oxidant sensitivity during development by measuring oxygen consumption using self-referencing oxygen sensing microelectrodes and reducing endogenous sources of ROS by over-expressing antioxidant enzymes on the outer mitochondrial membrane. Given the growing movement of government agencies and private industry to employ zebrafish for teratogenicity and developmental toxicity, it is especially important to comprehend the critical windows of sensitivity to oxidative stress and the underlying mechanisms. This research will provide a detailed and mechanistic understanding of how oxidative stress can impact embryonic development, and identify important considerations necessary when examining chemicals (and chemical mixtures that cause oxidative stress. It will also result in a better understanding of how oxidative stress influences the development and progression of human embryotoxicity and resulting teratogenesis and diseases.
|Wincent, Emma; Kubota, Akira; Timme-Laragy, Alicia et al. (2016) Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner. Biochem Pharmacol 110-111:117-29|
|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|
|Rousseau, Michelle E; Sant, Karilyn E; Borden, Linnea R et al. (2015) Regulation of Ahr signaling by Nrf2 during development: Effects of Nrf2a deficiency on PCB126 embryotoxicity in zebrafish (Danio rerio). Aquat Toxicol 167:157-71|
|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|
|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|
|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; 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|
|Timme-Laragy, Alicia R; Karchner, Sibel I; Hahn, Mark E (2012) Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology. Methods Mol Biol 889:51-71|
|Jonsson, Maria E; Kubota, Akira; Timme-Laragy, Alicia R et al. (2012) Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish. Toxicol Appl Pharmacol 265:166-74|