Department of Ecology/Hydrobiology Research Institute, Jinan University, Guangzhou 510632, China.
Department of Ecology/Hydrobiology Research Institute, Jinan University, Guangzhou 510632, China; Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China.
Sci Total Environ. 2018 Mar;616-617:583-593. doi: 10.1016/j.scitotenv.2017.10.305.
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 to 500μgL) 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.
作为一种广泛使用的药物，双氯芬酸（DCF）最近已在水生环境中普遍检出，并且也有报道称 DCF 暴露对哺乳动物产生了一些负面影响。然而，关于其对鱼类等非靶标生物的潜在有害影响的研究仍需要更多的调查。在这项研究中，我们选择了中国南方普遍存在的小型淡水入侵物种——食蚊鱼（Gambusia affinis）作为测试生物。我们首先克隆了关键的核转录因子相关基因孕烷 X 受体（PXR）及其下游基因的部分序列，包括 P-糖蛋白（P-gp）、细胞色素 3A（CYP3A）、多药耐药蛋白 2（MRP2）、谷胱甘肽过氧化物酶（GPx）和硫氧还蛋白还原酶（TXR）。基于推导的氨基酸序列，构建了 PXR、CYP3A 和 MRP2 的系统发育树。系统发育树和blast 结果表明，G. affinis 与其他花鳉物种（如 Xiphophorus maculatus）具有高度相似性。然后，在环境相关浓度的 DCF（从 0.5μgL 到 500μgL）暴露下，测量了上述基因及其部分相关酶/蛋白活性的转录表达水平，暴露时间分别为 24h 和 168h。结果表明，在 DCF 暴露下，PXR、CYP3A、P-gp 和 TXR 的 mRNA 表达明显诱导，表现出明显的时间效应关系，随着暴露时间的延长而延长。就酶活性和蛋白质含量而言，与转录水平相比，没有观察到明显的变化。Western blot 结果显示，随着 DCF 浓度的增加，PXR 蛋白在 24h 时增加，而在 168h 时减少，在一定程度上表现出剂量效应关系。GPX 活性在 24h 和 168h 均持续诱导，与 GPX 基因的表现具有很好的一致性。GSSH/T-GSH 在所有处理中均增加。总之，DCF 对食蚊鱼 PXR 及其下游靶基因的表达有可检测的影响。
