Aquatic Toxicology 84 (2007) 142–152 Regulation of branchial zinc uptake by 1,25-(OH) 2 D 3 in rainbow trout and associated changes in expression of ZIP1 and ECaC Andong Qiu a,1 , Chris N. Glover b , Christer Hogstrand a,b,∗ a King’s College London, Nutritional Sciences Division, Department of Biochemistry, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK b National Institute of Nutrition and Seafood Research, Postboks 2029, Nordnes, 5817 Bergen, Norway Received 2 November 2006; received in revised form 21 February 2007; accepted 21 February 2007 Abstract Zinc is a vital micronutrient to all organisms, but is also a toxicant to aquatic species. It is therefore of importance to determine the mechanisms by which zinc uptake is modulated. In the present study, we investigated the regulatory effects of the vitamin D metabolite, 1,25-(OH) 2 D 3 , on branchial zinc influx in rainbow trout, Oncorhynchus mykiss. Our results showed that branchial zinc uptake in rainbow trout was stimulated 7 days after a single intraperitoneal injection of 1,25-(OH) 2 D 3 (0.01 g/g fish). To understand the molecular components of zinc uptake regulation by 1,25-(OH) 2 D 3 , a ZIP zinc transporter (OmSLC39A1) and a partial vitamin D receptor (OmVDR) were molecularly cloned from rainbow trout gill, and the transcriptional expression of OmSLC39A1, epithelial calcium channel (OmECaC) and OmVDR genes in the gill was subsequently analyzed in response to 1,25-(OH) 2 D 3 . OmECaC, OmSLC39A1 and OmVDR were all upregulated following treatment with 1,25-(OH) 2 D 3 , but the effect was observed at different time points. OmECaC expression was significantly increased by 1,25-(OH) 2 D 3 on Days 3 and 5 after the injection, and expression of OmVDR was stimulated on Day 5. There was also an increased abundance of OmSLC39A1 mRNA on Day 7 following the injection with 1,25-(OH) 2 D 3 , but given the late response the effect of 1,25-(OH) 2 D 3 on this gene might be indirect. The results from the present study provide strong evidence that administration of 1,25-(OH) 2 D 3 results in enhanced zinc uptake across rainbow trout gill and that this effect is associated with an increased expression of transporters that mediate zinc uptake. The implications of our findings, in terms of aquatic toxicology, are that vitamin D status influences zinc accumulation in gill and body of fish. © 2007 Elsevier B.V. All rights reserved. Keywords: Calcitriol; SLC39A1; ZIP; ECaC; TRPV5; TRPV6; VDR 1. Introduction Excessive zinc accumulation can be toxic and has been linked to deleterious effects, such as hypocalcaemia (Spry and Wood, 1985) and neurodegeneration (Frederickson and Bush, 2001). Furthermore, zinc is an environmental hazard, frequently violat- ing water quality criteria in Europe and the USA. Zinc deficiency can also be a serious dilemma for organisms. It has been found that even moderate zinc deficiency can cause problems including Abbreviations: ZIP, ZRT1-, IRT1-like protein; ECaC, epithelial calcium channel; ZnT, zinc transporter; MT, metallothionein; aa, amino acid; VDR, vitamin D receptor; VDRE, vitamin D responsive element ∗ Corresponding author. Tel.: +44 20 7848 4436; fax: +44 20 7848 4500. E-mail address: christer.hogstrand@kcl.ac.uk (C. Hogstrand). 1 Present address: Department of Molecular Pharmacology, Einstein Cancer Research Center, The Albert Einstein College of Medicine of Yeshiva University, Chanin 628, 1300 Morris Park Avenue, Bronx, NY 10461, USA. anaemia, loss of appetite, immune system defects, developmen- tal impairment and teratogenesis (Castillo-Duran and Weisstaub, 2003; Walker and Black, 2004). In fish, zinc is not only absorbed in the gut, but also across the gills and the latter pathway is of high significance in terms of zinc toxicity. Spry and Wood (1989) showed that calcium competitively inhibited zinc uptake across rainbow trout gill. Later, it was found that zinc was a competitive inhibitor for branchial calcium uptake, indicating that zinc and calcium may share a common uptake site in the gills (Hogstrand et al., 1994). The nature of this site was revealed by the discovery of epithe- lial calcium channels (ECaC aka TRPV5/6) in Fugu pufferfish (Takifugu rubripes), zebrafish (Danio rerio), and rainbow trout (Oncorhynchus mykiss; Qiu and Hogstrand, 2004; Qiu, 2004; Pan et al., 2005; Shahsavarani et al., 2006). In post-larval stages of fish, ECaC is highly expressed in the gill with low mRNA levels present in other tissues (Qiu and Hogstrand, 2004; Qiu, 2004; Shahsavarani et al., 2006). Functional characterization 0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.02.020