Diclofenac exposure alter the expression of PXR and its downstream target genes in mosquito fish (Gambusia affinis)

Abstract

As one of widely used drugs, Diclofenac (DCF) recently has been universally detected in aquatic environment and some negative effects derived from DCF exposure to mammals have been also reported. However, studies about its potential deleterious effects on non-target organisms like fish still require more investigation. In this study an ubiquitous small freshwater invader species in Southern of China, mosquito fish (Gambusia affinis), was employed as test organism. We firstly cloned the crucial partial sequences of nucleus transcriptional factor related genes pregnane X receptor (PXR) and its downstream genes, including P-glycoprotein (P-gp), cytochrome 3A (CYP3A), multidrug resistance protein 2 (MRP2), glutathione peroxidase (GPx) and thioredoxin reductase (TXR) in mosquito fish. The phylogenetic trees of PXR, CYP3A and MRP2 were constructed based on their deduced amino acids sequences, respectively. Phylogenetic trees and blast results showed a high similarity between G. affinis and other killifish species, such as Xiphophorus maculatus. The transcriptional expression of these genes mentioned above and partly related enzymes/proteins activities were then measured under the exposure of environmentally relevant concentrations of DCF (from 0.5μgL−1 to 500μgL−1) for 24h and 168h. Results showed that the mRNA expression of PXR, CYP3A, P-gp and TXR showed dramatic induction under DCF exposure, exhibiting an obvious time-effect relationship with the extend of exposure time. In terms of enzyme activity and protein content, no dramatic changes as in transcription were observed. Western blotting showed PXR protein increased at 24h but decreased at 168h with the increasing of DCF concentration, displaying a dose-effect relationship to some extent. GPX activity was continuously induced both at 24h and 168h, exhibiting a good consistency with the performance of GPX gene. GSSH/T-GSH increased in all treatments. Overall, DCF had traceable effects on the expression of PXR and its downstream target genes in mosquito fish.