Aquatic Toxicology 179 (2016) 143–150 Contents lists available at ScienceDirect Aquatic Toxicology j ourna l ho me pa ge: www.elsevier.com/locate/aquatox Molecular mechanisms of selenium-Induced spinal deformities in fish Allison Kupsco ∗ , Daniel Schlenk Environmental Toxicology Program and Department of Environmental Sciences, University of California-Riverside, Riverside, CA, United States a r t i c l e i n f o Article history: Received 26 July 2016 Received in revised form 30 August 2016 Accepted 1 September 2016 Available online 3 September 2016 Keywords: Selenomethionine Japanese medaka Unfolded protein response Oxidative stress Apoptosis Skeletogenesis a b s t r a c t Selenium toxicity to oviparous vertebrates is often attributed to selenomethionine (SeMet), which can biomagnify through maternal transfer. Although oxidative stress is implicated in SeMet toxicity, knowl- edge gaps remain in how SeMet causes characteristic spinal deformities. In the present study, we use the Japanese medaka (Oryzias latipes) model to investigate the role of oxidative stress, cell death, and the unfolded protein response (UPR) on skeletal gene expression and SeMet toxicity, linking localiza- tion of cellular effects to observed abnormalities. Medaka embryos were treated with 2.5 M or 5 M SeMet for 24 h at stage 25 (48 h post fertilization). Post treatment, embryos were separated into normal, deformed (mild, moderate or severe), or dead categories. Dichlorofluorescein staining demonstrated oxidative stress in tails of embryos with observable spinal malformations. Furthermore, acridine orange staining for apoptosis identified significantly more dead cells in tails of treated embryos. Gene expres- sion studies for the UPR suggest a potential role for CHOP (c/ebp homologous protein) induced apoptosis deformed embryos after 5 M SeMet, accompanied by a significant decrease in PDIA4 (protein disul- fide isomerase A4) and no change in Dnajb9 (ER DNA J Domain-Containing Protein 4). This expression was distinct from the UPR induced by well-studied ER stress inducer, tunicamycin, which robustly acti- vated CHOP, PDIA4 and Dnajb9. Finally, SeMet treatment significantly decreased transcripts of cartilage development, Sox9 (SRY box 9), while increasing Runx2 in deformed embryos, without altering Twist or Collagen 2a1. Results suggest that oxidative stress, the UPR and cell death play key roles in SeMet induced deformities and altered skeletal development factors. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Selenium (Se) is an essential micronutrient with a narrow mar- gin between essentiality and toxicity to oviparous vertebrates. Although Se is present naturally in soils, anthropogenic disturbance can release Se into waterways. Free waterborne Se is bioaccumu- lated at low trophic levels, and integrated into selenomethionine (SeMet), which is then incorporated non-specifically into proteins (Fan et al., 2002). Thus, vertebrate consumption of Se in the diet is often in the form of SeMet. SeMet can be maternally transferred in vitellogenin to developing embryos, where it exerts devel- opmental toxicity causing teratogenesis and mortality. Common abnormalities from Se include spinal deformities, such as lordosis, cranio-facial abnormalities, and fin deformities (Lemly, 1997). In particular, skeletal deformities are characteristic of Se toxicity. Speculations about the mechanism of toxicity have suggested a role for oxidative stress in Se embryo toxicity (Lavado et al., 2012; ∗ Corresponding author at: Geology 2460, Environmental Sciences, University of California-Riverside, 900 University Ave, Riverside, CA 92521, United States. E-mail address: akups001@ucr.edu (A. Kupsco). Palace et al., 2004; Misra et al., 2012; Arnold et al., 2016). However, most studies were performed with high concentrations of SeMet, only a few link oxidative stress to an adverse outcome, and none have showed it occurring at the sites of malformations. Further- more, other research suggests that oxidative stress is not the only molecular and cellular disturbance caused by SeMet (Kupsco and Schlenk, 2014). Oxidative stress has been linked to the unfolded protein response (UPR) in several studies (Cao and Kaufman, 2014). The UPR is an integrated stress response activated by an increase in unfolded proteins in the endoplasmic reticulum (ER), which causes ER stress. The response induces increases in protein folding capac- ity, translational attenuation, mRNA degradation, and proteolysis (Hetz, 2012). Activation of the UPR occurs when the master reg- ulatory chaperone BiP dissociates from the three branches, ATF6 (Activating transcription factor 6), PERK (PKR-like endoplasmic reticulum kinase) and IRE1 (Inositol requiring enzyme 1), each with some independent function. IRE1 is primarily responsible for degradation of mRNA and proteins. PERK is responsible for trans- lational attenuation and ATF6 for an increase in folding capacity (Hetz, 2012). If the stress remains uncorrected, the UPR will initiate apoptosis via C/EBP homologous protein (CHOP). Apoptosis is pro- http://dx.doi.org/10.1016/j.aquatox.2016.09.001 0166-445X/© 2016 Elsevier B.V. All rights reserved.