Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin-o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.
Bibliographical noteFunding Information:
We would like to thank Dr. Diane Nacci and Ms. Denise Champlin (U.S. Environmental Protection Agency) for providing us with killifish, spawning baskets and advice on spawning and in ovo EROD; Dr. Doug Harrison (University of Kentucky) for use of his fluorescent microscope and advice on filters; Dr. Hong Yan, Dr. Elizabeth Debski, Ms. Bing Zhao and Ms. N. Virginia Lintecum (University of Kentucky) for their help in preparing killifish histology slides; Dr. Michael Moore (Woods Hole Oceanographic Institution) for confirming the identity of the embryonic liver. This research was sponsored by the National Institute of Health (F31 ES05942-04) predoctoral minority fellowship (X. A.), the Superfund Basic Research Program (P42 ES10356 to R.D.) and an EPA STAR fellowship to D.W.
Copyright 2008 Elsevier B.V., All rights reserved.
- In ovo EROD
- In ovo ROS