Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro

Polybrominated diphenyl ethers (PBDEs) are environmentally pervasive flame retardants that have been linked with endocrine disruption in a variety of organisms. BDE-47, one of the most prevalent congeners found in aquatic environments, has recently been shown to inhibit crustacean molting, but little is known about the specific mechanism through which molt-inhibition occurs. This study examined whether the inhibitory effect on molting arises from the disruption of hormone signaling in the epidermis using the blue crab, Callinectes sapidus, as the model crustacean. First, we partially sequenced cDNA of N-acetyl-β-glucosaminidase (NAG) from the epidermis, a terminal enzyme in the molting hormone-signaling cascades that is commonly used as the biomarker for ecdysteroid signaling. This partial cDNA sequence was then used to create primers for quantification of NAG gene expression. Then, a new tissue culture technique was developed and dubbed the epidermis-with-exoskeleton (EWE) method, wherein epidermal tissue, along with the overlying exoskeleton, is immersed in a medium of physiologically relevant osmolarity. Using this EWE tissue culture method, we assessed the inducibility of NAG mRNA by 20-hydroxyecdysone (20-HE) in vitro. Exposures to 1 μM 20-HE were found to induce NAG mRNA at a significantly higher level than the control. Using NAG expression as a biomarker for ecdysteroid signaling, the effects of BDE-47 were measured. BDE-47 alone at 100 nM and a combination of 1 μM BDE-47 and 1 μM 20-HE were found to significantly increase NAG mRNA. A trend of increasing NAG gene expression in the binary BDE-47 exposure as compared to 1 μM BDE-47 and 1 μM 20-HE alone is suggestive of a synergistic effect of these two chemicals on ecdysteroid signaling in the cultured epidermis. Discussion on the mechanism for inhibition of crustacean molting by BDE-47 is presented.