Sensitivity and toxic mode of action of dietary organic and inorganic selenium in Atlantic salmon (Salmo salar)

Highlights

• Dietary inorganic or organic selenium levels ∼1–2 mgkg⁻¹ do not cause toxic responses.

• Inorganic Se, but not organic Se, affects the S-Adenosylmethionine (SAM) pathway.

• Dietary organic and inorganic Se causes oxidative stress and altered lipid metabolism.

Abstract

Depending on its chemical form, selenium (Se) is a trace element with a narrow range between requirement and toxicity for most vertebrates. Traditional endpoints of Se toxicity include reduced growth, feed intake, and oxidative stress, while more recent finding describe disturbance in fatty acid synthesis as underlying toxic mechanism. To investigate overall metabolic mode of toxic action, with emphasis on lipid metabolism, a wide scope metabolomics pathway profiling was performed on Atlantic salmon (Salmo salar) (572 ± 7 g) that were fed organic and inorganic Se fortified diets. Atlantic salmon were fed a low natural background organic Se diet (0.35 mg Se kg⁻¹, wet weight (WW)) fortified with inorganic sodium selenite or organic selenomethionine-yeast (SeMet-yeast) at two levels (∼ 1–2 or 15 mg kg⁻¹, WW), in triplicate for 3 months. Apparent adverse effects were assessed by growth, feed intake, oxidative stress as production of thiobarbituric acid-reactive substances (TBARS) and levels of tocopherols, as well as an overall metabolomic pathway assessment. Fish fed 15 mg kg⁻¹ selenite, but not 15 mg kg⁻¹ SeMet-yeast, showed reduced feed intake, reduced growth, increased liver TBARS and reduced liver tocopherol. Main metabolic pathways significantly affected by 15 mg kg⁻¹ selenite, and to a lesser extent 15 mg kg⁻¹ SeMet-yeast, were lipid catabolism, endocannabinoids synthesis, and oxidant/glutathione metabolism. Disturbance in lipid metabolism was reflected by depressed levels of free fatty acids, monoacylglycerols and diacylglycerols as well as endocannabinoids. Specific for selenite was the significant reduction of metabolites in the S-Adenosylmethionine (SAM) pathway, indicating a use of methyl donors that could be allied with excess Se excretion. Dietary Se levels to respectively 1.1 and 2.1 mg kg⁻¹ selenite and SeMet-yeast did not affect any of the above mentioned parameters. Apparent toxic mechanisms at higher Se levels (15 mg kg⁻¹) included oxidative stress and altered lipid metabolism for both inorganic and organic Se, with higher toxicity for inorganic Se.

Introduction

Selenium (Se) is a well-known essential trace element active as part of functional selenoproteins (Kryukov and Gladyshev, 2000) involved in physiological processes such as antioxidant defense (glutathione peroxidases)(Toppo et al., 2008) and thyroid homeostasis (deiodinases)(Schweizer and Steegborn, 2015). However, Se has a narrow range between its toxic and its beneficial effects (Han et al., 2010, Lee et al., 2016, Wang and Lovell, 1997). In the aquatic system, Se contamination occurs as result of natural geological processes and/or anthropogenic pollution such as mining, irrigation runoff, and fossil fuel waste (Janz, 2012, Lemly, 2002). Inorganic Se is biotransformed to organoselenides including selenomethionine (SeMet), which accumulates along the aquatic food chain causing dietary exposure to fish (Maier and Knight, 1994). The organic SeMet form has a higher ability to raise tissue Se levels than inorganic selenite, which is attributed to the difference in metabolism of SeMet compared to selenite (Fontagne-Dicharry et al., 2015, Godin et al., 2015, Rider et al., 2009). Selenomethionine can be directly and nonspecifically incorporated in any protein containing methionine, while selenite needs to react with glutathione (GSH) to form hydrogen selenide (H₂Se) before being incorporated in specific selenoproteins (Godin et al., 2015, Suzuki, 2005). At excess intake, both SeMet and hydrogen selenite can be excreted into urine after methylation in the liver by using s-Adenosylmethionine (SAM) as methyl donor (Suzuki, 2005).

In general, inorganic Se is considered more toxic than organic Se forms (Thiry et al., 2012). However, organic SeMet has a narrow window of requirement and toxicity has been observed for Nile tilapia (Oreochromis niloticus) (Lee et al., 2016).Juvenile rainbow trout (Oncorhynchus mykiss) appears to have threshold levels for chronic dietary SeMet toxicity that is in the same range as for dietary selenite (Hamilton, 2004, Vidal et al., 2005). In fish in general, one of the major Se toxicity symptoms is embryo/larval teratogenic deformities in wild and laboratory studies (Janz, 2012, Lemly, 2002), although in a chronically dietary SeMet exposure study with cutthroat trout (Oncorhynchus clarki bouvieri) no change in reproductive performance was seen and offspring did not show appreciable deformities (Hardy et al., 2010). Other adverse effects include reduced reproduction in fathead minnow (Pmephales-promelas) (Schultz and Hermanutz, 1990) and altered sex steroid hormone production in female rainbow trout (Wiseman et al., 2011a), impaired growth in channel catfish (Ictalurus punctatus) (Wang and Lovell, 1997) and energy storage capacity in juvenile fathead minnow (McPhee and Janz, 2014), reduced feed intake in white sturgeon (Acipenser transmontanus) (Zee et al., 2016a), altered swimming performance in fathead minnow (McPhee and Janz, 2014, Thomas et al., 2013), induction of stress hormones in goldfish (Carassius auratus) and rainbow trout, respectively (Choi et al., 2015, Wiseman et al., 2011b), or reduced neurological and immunological functions in gold fish (Choi et al., 2015).

Oxidative stress has been suggested as one of the main causes of Se toxicity (Lee et al., 2015) for both inorganic Se (Choi et al., 2015, Hauser-Davis et al., 2016, Miller et al., 2007) and organic Se forms (Han et al., 2010, Hursky and Pietrock, 2015, Palace et al., 2004). Selenium-mediated oxidative stress has been attributed to Se’s ability to oxidize thiols (Spallholz, 1994), removing (SH) sulfhydryl groups in protein formation that are essential to cellular oxidative processes (Maier and Knight, 1994), and/or metabolism of inorganic and organic Se that create Se metabolites that form reactive oxygen species (Misra et al., 2012, Misra and Niyogi, 2009, Palace et al., 2004). In spite of these mechanisms, which are probably common for vertebrates, oxidative stress was not believed to be a main driver of dietary SeMet toxicity in white sturgeon (Acipenser transmontanus) (Zee et al., 2016a, Zee et al., 2016b). In least killifish (Heterandria formosa) dietary selenite and SeMet reduced rather than increased oxidative stress, but at the same time inhibited osmoregulatory enzymes, (Xie et al., 2016). Furthermore, in juvenile rainbow trout exposed to selenized-yeast worms, no oxidative stress response were seen. However, growth and liver triglyceride (TAG) levels were reduced (Knight et al., 2016). Recently, several wide-scope pathway assessments have shown that disturbed lipid synthesis and metabolism could be one of the main drivers of organic Se toxicity in rainbow trout (Knight et al., 2016, Pacitti et al., 2016a). Transcriptomic approaches to determine the underlying mechanisms associated with dietary Se toxicity in juvenile rainbow trout, showed that organic Se increases the expression of networks for growth-related signaling cascades in addition to those related to fatty acid synthesis and metabolism (Knight et al., 2016). The disruption of metabolites related to triglyceride processing and storage, as well as gene networks for epidermal growth factor and Notch signaling in the liver, were suggested to represent key molecular initiating events for adverse outcomes related to growth and Se toxicity in fish (Knight et al., 2016). Other transcriptomic assessments of rainbow trout fed Se-yeast, showed that in liver, lipid metabolism was the main pathway altered by Se exposures (Pacitti et al., 2016a). The use of wide-scope “omic” tools has been proven to be valuable tool in identifying new pathways of toxic responses, identifying possible new biomarkers of toxicity in addition to already identified classic end-point of toxicity (Shaw, 2006). In addition to transcriptomics, proteomics (profiling of proteins) and metabolomics (profiling of biochemicals or metabolites) are used to identify possible biomarkers of toxicity (Shaw, 2006). Metabolomics have been used to identify pathways of toxicity for several contaminants in in vitro (Olsvik et al., 2015, Softeland et al., 2014) and in vivo fish trials (Berntssen et al., 2016), and have been used to assess Se toxicity in yeast (Kitajima et al., 2012) and mammals (Garcia-Sevillano et al., 2013).

In Atlantic salmon, little is known about the toxic mode of action of Se. Thus, the purpose of the present study was to assess the underlying toxic mechanisms and sensitivity of both dietary selenite and SeMet-yeast in Atlantic salmon by using classic endpoints of Se toxicity such as growth, oxidative stress as well as overall metabolomics profiling approaches to assess non-target endpoints of toxicity. Establishing the sensitivity of Atlantic salmon to dietary selenite and SeMet-yeast dose has implications for setting safe limits in aquatic environments such as threshold body and tissue levels.