Aquatic Toxicology 164 (2015) 16–22 Contents lists available at ScienceDirect Aquatic Toxicology j o ur na l ho me pag e: www.elsevier.com/locate/aquatox Microcystin-RR exposure results in growth impairment by disrupting thyroid endocrine in zebrafish larvae Liqiang Xie a , Wei Yan b , Jing Li c , Liqin Yu c , Jianghua Wang c , Guangyu Li c,d,∗ , Nan Chen c,∗ , Alan D. Steinman e a State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, 73 East Beijing Road, Nanjing 210008, China b Institute of Agricultural Quality Standards & Testing Technology, Hubei Academy of Agricultural Sciences, Wuhan 430064, China c College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China d Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China e Robert B. Annis Water Resources Institute, Grand Valley State University, 740 West Shoreline Drive, Muskegon, MI 49441, USA a r t i c l e i n f o Article history: Received 25 November 2014 Received in revised form 8 April 2015 Accepted 10 April 2015 Available online 13 April 2015 Keywords: MC-RR Developing zebrafish Growth impairment Hypothalamic–pituitary–thyroid axis a b s t r a c t Recent studies have shown that cyanobacteria-derived microcystins (MCs) have the potential to disrupt endocrine systems. However, the effects of microcystin-RR (MC-RR) and their underlying mechanisms are poorly resolved in fish. In this study, MC-RR exposure through submersion caused serious developmental toxicity, such as growth delay and depressed heart rates in zebrafish larvae. We also detected decreased levels of thyroid hormones (THs), suggesting that MC-RR-triggered thyroid endocrine disruption might contribute to the growth impairment observed in developing zebrafish. To further our understanding of mechanisms of MC-RR-induced endocrine toxicity, quantitative real-time PCR (QPCR) analysis was per- formed on hypothalamic–pituitary–thyroid (HPT) axis related genes, i.e., corticotropin-releasing factor (CRF), thyroid-stimulating hormone (TSH), sodium/iodide symporter (NIS), thyroglobulin (TG), thyroid receptors (TR and TR) and iodothyronine deiodinases (Dio1 and Dio2), of developing zebrafish embryos exposed to 0, 0.3, 1.0 or 3.0 mg L −1 MC-RR until 96 h post-fertilization. Our results showed that tran- scription pattern of HPT axis related genes were greatly changed by MC-RR exposure, except TG gene. Furthermore, western blot was used to validate the results of gene expression. The results showed protein synthesis of TG was not affected, while that of NIS was significantly up-regulated, which are in accordance with gene expression. The overall results indicated that exposure to MC-RR can induce developmental toxicity, which might be associated with thyroid endocrine disruption in developing zebrafish larvae. © 2015 Elsevier B.V. All rights reserved. 1. Introduction It is widely accepted that the frequency and global distribution of toxic cyanobacterial blooms in aquatic systems have signifi- cantly increased and become a worldwide concern in recent years. Among cyanotoxins, microcystins (MCs) are a group of toxic cyclic heptapeptides with more than 100 different structural variants (Kaasalainen et al., 2012), among which microcystin-RR (MC-RR) is one of the dominant variants in cyanobacterial blooms in lakes of China (Xie et al., 2005; Chen et al., 2006). ∗ Corresponding authors at: College of Fisheries, Huazhong Agricultural Univer- sity, Wuhan 430070, China. Fax: +86 27 87282114. E-mail addresses: ligy2001@163.com (G. Li), chennan@mail.hzau.edu.cn (N. Chen). MCs are generally associated with hepatotoxicity. The typical toxicological action of MCs is to inhibit serine/threonine protein phosphatase (Eriksson et al., 1990; Falconer and Yeung, 1992). Additionally, MCs have been reported to induce both production of reactive oxygen species (ROS) (Ding et al., 2000, 2001) and destruction of the cytoskeleton, eventually leading to liver necrosis, apoptosis and hemorrhage (Fischer and Dietrich, 2000; Gehringer, 2004). Besides the liver, MCs were found to accumulate and cause damage in the kidney, heart, brain and gonad of embryos and adults (Zhang et al., 2008; Qiu et al., 2009; Li et al., 2011; Zeng et al., 2014). Recently, the endocrine-disrupting effect of MCs has attracted much more public concern. Both laboratory and field studies have discovered that MCs can disrupt the endocrine systems in mam- mals and fish. It was reported that injection with microcystin-LR (MC-LR) can affect male mice serum hormones and mRNA expres- sions by damaging the hypothalamic–pituitary systems (Wang http://dx.doi.org/10.1016/j.aquatox.2015.04.014 0166-445X/© 2015 Elsevier B.V. All rights reserved.