Effects of triaryl phosphates on mouse and human nuclear receptors Paavo Honkakoski a,* , Jorma J. Palvimo b , Leena Penttila ¨ c , Jouko Vepsa ¨la ¨inen d , Seppo Auriola c a Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland b Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FIN-00014, Helsinki, Finland c Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland d Department of Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland Received 13 June 2003; accepted 25 August 2003 Abstract The constitutively active receptor (CAR) is a crucial regulator of genes encoding for enzymes active in drug/steroid oxidation, conjugation, and transport. In our attempt to isolate the endogenous inhibitory ligand(s) for the mouse CAR, we found surprisingly that the inhibitory activity was associated with di- and tri-isopropylated phenyl phosphates that were present in livers of untreated mice. Trans- activation experiments in mammalian cells with synthetic compounds verified that mouse CAR was inhibited by various isopropylated phenyl phosphates (40–80%). Such triaryl phosphates are widely used as fire retardants, lubricants, and plasticizers, and some of them are known to disturb reproduction by currently unknown mechanisms. Equipped with the finding that these compounds could interact with mouse CAR, we proceeded to determine their functional effects on other nuclear receptors. Human CAR and pregnane X receptor (PXR) were variably activated (2–5-fold) by triaryl phosphates while mouse PXR, peroxisome proliferator-activated receptor-a, and vitamin D receptor were refractory. Among steroid hormone receptors, the human androgen receptor was inhibited by triphenyl phosphate and di-ortho-isopropylated phenyl phosphate (40–50%) and activated by di- and tri-para-substituted phenyl phosphates (2-fold). Our results add to the list of CAR and PXR activators and suggest steroid-dependent biological pathways that may contribute to the reproductive effects of triaryl phosphates. # 2003 Elsevier Inc. All rights reserved. Keywords: Organophosphates; Triaryl phosphates; Nuclear receptor; Activation; Mouse; Human 1. Introduction NRs are ligand-dependent DNA-binding transcription factors that are encoded by a superfamily of 48 genes [1]. The NRs transmit extra- or intracellular signals directly to gene transcription machinery, in response to steroid and thyroid hormones, fatty acid and cholesterol derivatives, and vitamins A and D. NRs control profound cellular processes such as growth and differentiation, carbohydrate and lipid metabolism, and endocrine physiology [2]. Therefore, dis- turbances in function of NRs may result in clinical mani- festations. Such disturbances include hormone resistance syndromes and cancers where interindividual differences in NR structure result in abnormal ligand binding or changes in responses to NR co-regulators [3,4]. On the other hand, environmental or diet-derived chemicals (xenobiotics) may disturb hormonal balance via binding to NRs [5–7]. Xenobiotics may also affect hormonal balance through activation of CYP gene expression. CYPs are essential not only in the metabolism of xenobiotics but also in the control of both formation and degradation of endogenous compounds (endobiotics) which include NR ligands [8]. Because CYP genes are regulated by NRs [9], exposure to xenobiotics may lead to increased expression of CYP enzymes and enhanced xeno- and endobiotic metabolism. While the ligand specificities of steroid hormone recep- tors have been well established, many of the so-called Biochemical Pharmacology 67 (2004) 97–106 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2003.08.037 * Corresponding author. Tel.: þ358-17-162490; fax: þ358-17-162252. E-mail address: paavo.honkakoski@uku.fi (P. Honkakoski). Abbreviations: AR, androgen receptor; CAR, constitutively active receptor; CYP, cytochrome P450; ERa, estrogen receptor-a; GR, glucocorticoid receptor; NR, nuclear receptor; PPARa, peroxisome proliferator-activated receptor-a; PR, progesterone receptor; PXR, preg- nane X receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; TPP, triphenyl phosphate; TTP, tri-p-methyl phenyl phosphate. Isopropy- lated triaryl phosphates are abbreviated by the generic symbol o-, m-, p- iPrXN, where o-, m-, and p- denote the position of the isopropyl (iPr) substituent on the phenyl moiety, and XN denotes the number of isopropylated phenyl moieties (N ¼ 1, 2, or 3).