Selenoneine, a Novel Selenium-Containing Compound, Mediates Detoxification Mechanisms against Methylmercury Accumulation and Toxicity in Zebrafish Embryo

Michiaki Yamashita 0 Yumiko Yamashita 0 Tamami Suzuki 0 Yoko Kani 0 Nanami Mizusawa 0 Shintaro Imamura 0 Kenji Takemoto 0 Tatsuro Hara 0 Md. Anwar Hossain 0 Takeshi Yabu 0 Ken Touhata 0 0 Present Address: T. Yabu Nihon University , 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan The selenium (Se)-containing antioxidant selenoneine (2-selenyl-N,N,N-trimethyl-L-histidine) has recently been discovered to be the predominant form of organic Se in tuna blood. Although dietary intake of fish Se has been suggested to reduce methylmercury (MeHg) toxicity, the molecular mechanism of MeHg detoxification by Se has not yet been determined. Here, we report evidence that selenoneine accelerates the excretion and demethylation of MeHg, mediated by a selenoneine-specific transporter, organic cations/carnitine transporter-1 (OCTN1). Selenoneine was incorporated into human embryonic kidney HEK293 cells transiently overexpressing OCTN1 and zebrafish blood cells by OCTN1. The Km for selenoneine uptake was 13.0 M in OCTN1-overexpressing HEK293 cells and 9.5 M in zebrafish blood cells, indicating high affinity of OCTN1 for selenoneine in human and zebrafish cells. When such OCTN1-expressing cells and embryos were exposed to MeHg-cysteine (MeHgCys), MeHg accumulation was decreased and the excretion and demethylation of MeHg were enhanced by selenoneine. In addition, exosomal secretion vesicles were detected in the culture water of embryos that had been microinjected with MeHgCys, suggesting that these may be responsible for MeHg excretion and demethylation. In contrast, OCTN1-deficient embryos accumulated MeHg, and MeHg excretion and demethylation were decreased. Furthermore, Hg accumulation was decreased in OCTN1-overexpressing HEK293 cells, but not in mock vector-transfected cells, indicating that selenoneine and OCTN1 can regulate MeHg detoxification in human cells. Thus, the selenoneine-mediated OCTN1 system regulates secretory lysosomal vesicle formation and MeHg demethylation. - Dietary intake of selenium (Se), which is found in fish, can reduce methylmercury (MeHg) toxicity (Friedman et al. 1978; Ganther and Sunde 1974; Ganther et al. 1972; Ohi et al. 1980; Raymond and Ralston 2004; Raymond et al. 2012). Ganther et al. (1972) demonstrated that intake of canned tuna meat containing slightly elevated MeHg levels reduced the toxicity to Japanese quail and suggested the protective role of Se. Friedman et al. (1978) showed a protective effect of freezedried swordfish meat against MeHg toxicity in rats, and Ohi et al. (1980) suggested that an unknown organic Se compound in fish meat could reduce MeHg neurotoxicity. Recently, a novel Se compound, selenoneine, was identified in the blood of bluefin tuna and distributed in fish muscles (Yamashita and Yamashita 2010; Yamashita et al. 2010; 2011). The generation of reactive oxygen species (ROS) has been linked to MeHg-induced toxicity both in vivo and in vitro (Petroni et al. 2012; Yin et al. 2007; Usuki et al. 2011), and selenoneine has strong radical scavenging properties (Yamashita and Yamashita 2010; Yamashita et al. 2010, 2011). Thus, selenoneine is proposed to be a biologically and nutritionally active chemical with the ability to detoxify MeHg in animal cells and tissues. With one Se atom in its imidazole ring, selenoneine is a Se analog of ergothioneine (Yamashita and Yamashita 2010). The organic cations/carnitine transporter, OCTN1 [solute carrier family 22, member 4; SLC22A4], transports ergothioneine and carnitine (Grndemann et al. 2005; Tamai et al. 1999) and therefore may also be involved in the uptake of selenoneine and its detoxification of MeHg. OCTN1 is ubiquitously distributed in mammalian cells and tissues, with erythrocytes having the highest OCTN1 mRNA levels (Grndemann et al. 2005; Tamai et al. 1999). Human embryonic kidney 293 (HEK293) cells transfected with an OCTN1 expression vector showed specific uptake of ergothioneine (Grndemann et al. 2005). The slc22a4 transporter gene has also been identified and expressed in zebrafish blood cells and other cell types, as expected given its role in heme metabolism (Nilsson et al. 2009; Weber et al. 2005). To identify the MeHg excretion pathway mediated by the selenium redox function, we focused on the ceramidemediated exosome secretion pathway. It has been shown recently that the formation and release of exosomes (small membrane-bound vesicles) by the endosomal sorting complex required for transport (ESCRT) machinery is triggered by ceramide generation (Trajkovic et al. 2008). Ceramide is produced by the hydrolytic action of sphingomyelinase (SMase) on substrate sphingomyelin in response to various agonists or stresses such as irradiation, heat shock, and oxidative stress (Hannun 1996; Mathias et al. 1993; Okazaki et al. 1989; Verheij et al. 1996; Yabu et al. 2008). The sphingomyelin pathway may be linked to stressinducible apoptosis and detoxification under oxidative stress conditions produced by MeHg exposure. Inorganic mercury (Hg) is distributed in marlin meat and in the livers and kidneys of fish and laboratory animals chronically exposed to MeHg (Clarkson 1972; Friberg and Mottet 1989; Itano et al. 1985; Ng et al. 2001; Nigro and Leonzio 1996; Palmisano et al. 1995; Skerfving 1978). Although demethylation of MeHg is thought to be an important route of detoxification, the underlying molecular mechanism is poorly understood. Therefore, the effects of chronic exposure to low MeHg levels should be characterized in animal models. Bioaccumulation and detoxification of heavy metals in aquatic and marine organisms are important research topics in the field of marine biotechnology (Auslander et al. 2008; Cordeiro et al. 2012; De et al. 2008; Ma et al. 2012). The zebrafish is a useful model organism for studying embryonic development and is increasingly being used in vertebrate toxicology studies (Gonzalez et al. 2005; Korbas et al. 2008; Kusik et al. 2008; Samson et al. 2001; Weber et al. 2008). Previously, embryonic and larval zebrafish have been used to study the toxic effects of MeHg exposure on the entire body (Samson et al. 2001; Weber et al. 2008) and at the molecular level (Gonzalez et al. 2005; Korbas et al. 2008). Because the nucleotide sequence and expression of the zebrafish OCTN1 gene homolog has been characterized (Nilsson et al. 2009; Weber et al. 2005), OCTN1-deficient embryos can be generated by microinjection of an antisense morpholino oligo (MO) and used for the study of MeHg toxicity. In the present study, we investigated the involvement of selenoneine and OCTN1 in MeHg detoxification using the zebrafish embryo model. The specific incorporation of selenoneine through OCTN1 was characterized in zebrafish embryos. In addition, the incorporation of selenoneine through the human homolog of OCTN1 was characterized in HEK293 cells. Morphological defects and apoptosis induced by MeHg were decreased by selenoneine exposure, and selenoneine reduced MeHg accumulati (...truncated)