Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

Abstract

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR22Zebrafish cytochrome P450 family 1 genes/mRNAs and proteins are referred to as CYP1 and CYP1 according to Nelson et al. (1996). For other genes/mRNAs and proteins in zebrafish we have followed the approved guidelines for zebrafish, e.g., ahr2 and Ahr2 (https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines). The aryl hydrocarbon receptor is denoted AHR when not referring to a particular species.) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0nM for induction of the CYP1s, 3.7 and 5.1nM (or higher) for cox-2a and cox-2b induction, and 2.5nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos33Fish embryos that have hatched but are still dependent on yolk as a nutrition source are technically “eleutheroembryos”, but for simplicity we will refer to them generically as “embryos” for the remainder of this paper. Once independent feeding begins (days 6–7 post fertilization in zebrafish), the fish are then called larvae. failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.