Arachidonic acid-containing phosphatidylcholine characterized by consolidated plasma and liver lipidomics as an early onset marker for tamoxifen-induced hepatic phospholipidosis Kosuke Saito a† , Keisuke Goda b† , Akio Kobayashi b *, Naohito Yamada b , Kyoko Maekawa a , Yoshiro Saito a * and Shoichiro Sugai b ABSTRACT: Lipid profiling has emerged as an effective approach to not only screen disease and drug toxicity biomarkers but also understand their underlying mechanisms of action. Tamoxifen, a widely used antiestrogenic agent for adjuvant therapy against estrogen-positive breast cancer, possesses side effects such as hepatic steatosis and phospholipidosis (PLD). In the present study, we administered tamoxifen to Sprague–Dawley rats and used lipidomics to reveal tamoxifen-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. Treatment with tamoxifen for 28days caused hepatic PLD in rats. We compared the plasma and liver lipid profiles in treated vs. untreated rats using a multivariate analysis to determine differences between the two groups. In total, 25 plasma and 45 liver lipids were identified and altered in the tamoxifen- treated group. Of these lipids, arachidonic acid (AA)-containing phosphatidylcholines (PCs), such as PC (17:0/20:4) and PC (18:1/20:4), were commonly reduced in both plasma and liver. Conversely, tamoxifen increased other phosphoglycerolipids in the liver, such as phosphatidylethanolamine (18:1/18:1) and phosphatidylinositol (18:0/18:2). We also examined alteration of AA-containing PCs and some phosphoglycerolipids in the pre-PLD stage and found that these lipid alterations were initiated be- fore pathological alteration in the liver. In addition, changes in plasma and liver levels of AA-containing PCs were linearly associated. Moreover, levels of free AA and mRNA levels of AA-synthesizing enzymes, such as fatty acid desaturase 1 and 2, were decreased by tamoxifen treatment. Therefore, our study demonstrated that AA-containing PCs might have potential utility as novel and predictive biomarkers for tamoxifen-induced PLD. Copyright © 2017 John Wiley & Sons, Ltd. Keywords: Lipidomics; biomarker; phospholipidosis; tamoxifen; plasma–liver association Introduction Lipids, a class of biological molecules, consist of several categories that include phosphoglycerolipids, sphingolipids and neutral lipids, which are components of cellular membranes and blood- circulating lipoproteins. Consequently, their levels in blood and organs, particularly liver lipids, are thought to be closely correlated (Kotronen et al., 2010). In addition, these lipids also play a pivotal role in various biological processes such as cellular proliferation, apoptosis and inflammation (Blumberg et al., 1995; Hannun and Linardic, 1993; Mené et al., 1989; Pettus et al., 2004). Therefore, blood lipid profiling would be an effective approach to screen and/or identify biomarkers for not only diseases but also drug toxicity. Lipidomics, a mass spectrometry-based approach for simultaneous assay of lipid molecules in biological samples such as blood and organs (Han and Gross, 2003; Houjou et al., 2005) has been used to identify drug toxicity biomarkers in blood. For example, lysophosphatidylcholines (LPC), such as LPC (20:4), in serum have been shown to be biomarkers for acetaminophen- and D-galactosamine-induced liver injuries (Cheng et al., 2009; Ma et al., 2014). Furthermore, bis-monophosphatidic acid (BMP) in serum and glycosylceramide (GCer) and LPC in plasma were shown to be markers for drug-induced hepatic phospholipidosis (PLD) (Mortuza et al., 2003; Saito et al., 2014). However, it remains possible that the altered biomarker levels are a consequence of off-target effects on organs other than liver by the toxicants/drugs. Although there is an association between the static lipid levels in serum/plasma and liver (Kotronen et al., 2010), data correlating serum/plasma–liver lipid levels with liver toxicity remain limited. Tamoxifen is a widely used antiestrogenic agent for adjuvant therapy in estrogen-positive breast cancer (Early Breast Cancer Trialists’ Collaborative Group, 1998; Jordan, 1988). Although tamoxifen is frequently used and is clinically effective, it induces side *Correspondence to: Akio Kobayashi, Toxicology Research Lab., Central Pharmaceu- tical Research Institute, Japan Tobacco Inc., 23 Naganuki, Hadano, Kanagawa 257-0024, Japan. E-mail: akio.kobayashi@jt.com Yoshiro Saito, Division of Medicinal Safety Science, National Institute of Health Sci- ences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. E-mail: yoshiro@nihs.go.jp † Authors contributed equally. a Division of Medicinal Safety Science, National Institute of Health Sciences, Setagaya, Tokyo, 158-8501, Japan b Toxicology Research Lab, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Hadano, Kanagawa, 257-0024, Japan J. Appl. Toxicol. 2017 Copyright © 2017 John Wiley & Sons, Ltd. Research article Received: 10 November 2016, Revised: 16 December 2016, Accepted: 26 December 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.3442