Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes Masato Honda a , Akemi Muta a , Taiki Akasaka b , Yoshiyuki Inoue c , Yohei Shimasaki a , Kurunthachalam Kannan d , Nozomu Okino e , Yuji Oshima a,n a Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan b Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan c Chemical Biotesting Center, Chemicals Evaluation and Research Institute, Bunkyo-ku, Tokyo 112-0004, Japan d Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY 12201-0509, USA e Laboratory of Marine Resource Chemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan article info Article history: Received 17 September 2013 Received in revised form 12 November 2013 Accepted 17 November 2013 Available online 11 March 2014 Keywords: Perfluorooctane sulfonate Plasma Binding protein Pufferfish Apolipoprotein A–I abstract It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190 ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies. & 2013 Elsevier Inc. All rights reserved. 1. Introduction Because of the high energy carbon-fluorine bond and amphi- pathic property obtained from hydrophilic (–SO 3 H) and hydropho- bic (CF 3 –(CF 2 ) 7 –) moieties, perfluorooctane sulfonate (C 8 HF 17 O 3 S, PFOS) is a stable compound and possesses superior properties such as chemical and abrasion resistance, and surfactant characteristics (Giesy and Kannan, 2002). In industrial and commercial applica- tions, PFOS is widely used all over the world as a mist suppressant, leveling agent, aqueous film forming foam, surfactant, and coating agent (Giesy and Kannan, 2002). Following widespread use for over five decades, PFOS was identified as a global environmental pollutant in 2001 (Giesy and Kannan, 2001; Harada and Koizumi, 2009; Taniyasu et al., 2003). Several studies have reported that PFOS is widely distributed in seawater, fish, sea birds, marine mammals, and polar bears (Bossi et al., 2005; Hölzer et al., 2011; Houde et al., 2011; Smithwick et al., 2005; Yamashita et al., 2008). PFOS is also an ubiquitous contaminant in human blood (Kannan et al., 2004; Olsen et al., 2003; Zhao et al., 2012). In 2009, PFOS was banned or regulated in many countries, and was designated as a persistent organic pollutant (POP) by the Stockholm Convention (Lindstrom et al., 2011). In Japan, PFOS was banned from industrial use in 2009. However, several countries continue to use PFOS and because of its persistent and bioaccumu- lative properties, its harmful effects as an environmental pollutant will continue in the near future (Zareitalabad et al., 2013). PFOS concentrations in wildlife, especially fish, are higher in plasma and liver than in other tissues (Martin et al., 2003; Ahrens et al., 2009). For example, in rainbow trout, Oncorhynchus mykiss, PFOS accumulated in blood at higher levels than in liver and other tissues (Martin et al., 2003); in Japanese scad, Trachurus japonicas, the mean PFOS concentrations were 170 ng/mL and 9 ng/g in blood and liver, respectively (Taniyasu et al., 2003). Reports from the National Health and Nutrition Examination Survey (NHANES) of the United States indicate that PFOS was detected in human serum at concentrations ranging from below the limit of detection to 435 ng/mL, and PFOS was detected in 99.9 percent of 2094 specimens analyzed (Calafat et al., 2007). In rats, urinary and fecal Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ecoenv Ecotoxicology and Environmental Safety 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.11.010 n Corresponding author. Fax: þ81 92 642 2904. E-mail address: yoshima@agr.kyushu-u.ac.jp (Y. Oshima). Ecotoxicology and Environmental Safety 104 (2014) 409–413