ABSTRACT
Exposures to co-planar PCBs and dioxins have been associated with diabetes in epidemiologic studies. Individuals may be predisposed to diseases such as diabetes as a result of exposure to environmental contaminants during early life, resulting in dysmorphic pancreatic islets or metabolically fragile ß-cells. We tested the hypothesis that embryonic exposure to a model Ahr-ligand, PCB-126 would cause structural and/or functional alterations to the developing primary pancreatic islet in the zebrafish (Danio rerio). To assess ß-cell development, transgenic zebrafish embryos (Tg(ins:GFP) and Tg(ins:mcherry) were exposed to nominal concentrations of 2 or 5nM PCB-126 or DMSO from 24-48h post fertilization (hpf), and imaged via time-lapse microscopy from 80-102hpf. We identified defects including hypomorphic islets, altered islet migration, islet fragmentation, and formation of ectopic ß-cells. As we recently showed the transcription factor Nrf2a is protective in PCB-126 embryotoxicity, we then assessed the transcriptional function of the islets in wildtype and nrf2a(fh318/fh318) mutant embryos. We measured gene expression of preproinsulin a, somatostatin2, pdx1, ghrelin, and glucagon. Expression of preproinsulin a increased with PCB treatment in wildtype embryos, while expression of all measured pancreas genes was altered by the nrf2a mutant genotype, suggesting misregulation of the glucose homeostasis axis in those embryos, independent of PCB treatment. This study shows that embryonic exposure to PCB-126 can result in deviant development of the pancreatic islet and suggests that Nrf2a plays a role in regulating glucose homeostasis during development.
Subject(s)
Insulin-Secreting Cells , Polychlorinated Biphenyls , Zebrafish , Animals , Animals, Genetically Modified/embryology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/embryology , Gene Expression Regulation, Developmental/drug effects , Ghrelin/genetics , Glucagon/genetics , Homeodomain Proteins/genetics , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/physiology , NF-E2-Related Factor 2 , Polychlorinated Biphenyls/adverse effects , Trans-Activators/genetics , Transcription Factors/genetics , Zebrafish/embryology , Zebrafish Proteins/geneticsABSTRACT
The embryotoxicity of co-planar PCBs is regulated by the aryl hydrocarbon receptor (Ahr), and has been reported to involve oxidative stress. Ahr participates in crosstalk with another transcription factor, Nfe2l2, or Nrf2. Nrf2 binds to antioxidant response elements to regulate the adaptive response to oxidative stress. To explore aspects of the crosstalk between Nrf2 and Ahr and its impact on development, we used zebrafish (Danio rerio) with a mutated DNA binding domain in Nrf2a (nrf2a(fh318/fh318)), rendering these embryos more sensitive to oxidative stress. Embryos were exposed to 2 nM or 5 nM PCB126 at 24 h post fertilization (prim-5 stage of pharyngula) and examined for gene expression and morphology at 4 days post fertilization (dpf; protruding - mouth stage). Nrf2a mutant eleutheroembryos were more sensitive to PCB126 toxicity at 4 dpf, and in the absence of treatment also displayed some subtle developmental differences from wildtype embryos, including delayed inflation of the swim bladder and smaller yolk sacs. We used qPCR to measure changes in expression of the nrf gene family, keap1a, keap1b, the ahr gene family, and known target genes. cyp1a induction by PCB126 was enhanced in the Nrf2a mutants (156-fold in wildtypes vs. 228-fold in mutants exposed to 5 nM). Decreased expression of heme oxygenase (decycling) 1 (hmox1) in the Nrf2a mutants was accompanied by increased nrf2b expression. Target genes of Nrf2a and AhR2, NAD(P)H:quinone oxidoreductase 1 (nqo1) and glutathione S-transferase, alpha-like (gsta1), showed a 2-5-fold increase in expression in the Nrf2a mutants as compared to wildtype. This study elucidates the interaction between two important transcription factor pathways in the developmental toxicity of co-planar PCBs.
