ACCELERATED COMMUNICATION Di(2-ethylhexyl) phthalate Is a Highly Potent Agonist for the Human Constitutive Androstane Receptor Splice Variant CAR2

The human constitutive androstane receptor (CAR, CAR1) regulates the expression of genes involved in xenobiotic metabolism in the liver. The CAR gene uses multiple alternative splicing events during pre-mRNA processing, thereby enhancing the CAR transcriptome. Previous reports have identified two prominent human CAR variants, CAR2 and CAR3, that possess fourand five-amino acid insertions in their ligand binding domains, respectively. Unlike the constitutively active reference form of the receptor, we now demonstrate that CAR2 is a ligandactivated receptor and comprises approximately 30% of the reference transcript level in human liver tissues in human hepatocytes. Furthermore, we identify the common plasticizer, di(2ethylhexyl) phthalate (DEHP), as a highly potent and uniquely selective agonist of CAR2. Results from reporter transactivation and mammalian two-hybrid assays reveal that DEHP activates CAR2 at low nanomolar concentrations, results further supported by analysis of CAR target gene expression in primary human hepatocytes. In addition, comparative genomic analyses show that the typical mouse, rat, and marmoset models of DEHP toxicity cannot accurately profile potential human toxicity because of these species’ inability to generate a CAR2-like transcript. The discovery that CAR2 is an ultimate human DEHP receptor identifies a novel pathway modulating human DEHP toxicity with potential clinical implications for a subset of patients undergoing critical care medical interventions. The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor superfamily that is expressed primarily in the liver (Baes et al., 1994). In conjunction with the pregnane X receptor (NR1I2), CAR regulates hepatic genes involved in all three phases of xenobiotic metabolism (Wei et al., 2000). The products of these genes play a role in modulating the metabolism of a variety of pharmaceuticals, such as acetaminophen (Zhang et al., 2002) and cyclophosphamide (Roy et al., 1999), as well as endogenous compounds, including thyroid hormone (Maglich et al., 2004), bile acids (Guo et al., 2003), and steroid hormones (Xie et al., 2003). In human livers, the CAR gene expresses a number of differentially spliced mRNA transcripts, generated through the use of alternative splice acceptor sites during pre-mRNA processing (Auerbach et al., 2003; Savkur et al., 2003; Arnold et al., 2004; Jinno et al., 2004; Lamba et al., 2004). Previous reports from our laboratory have focused on functional analysis of two prominent human CAR variants, termed CAR2 (Auerbach et al., 2007) and CAR3 (Auerbach et al., 2005). This work was supported, in part, by the National Institutes of Health National Institute of General Medical Sciences [Grant GM66411]; by the Intramural Research program of the National Institutes of Health National Institute of Environmental Health Sciences; and by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Contract N01-DK-7-0004/HHSN267200700004c]. The Quattro Premier XE mass spectrometer was purchased with funds from the National Science Foundation [Grant DBI-0619489]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.053702. ABBREVIATIONS: CAR, constitutive androstane receptor; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime; DEHP, di(2-ethylhexyl) phthalate; DMSO, dimethyl sulfoxide; RXR, retinoid X receptor; CMV, cytomegalovirus; FBS, fetal bovine serum; SRC1, steroid receptor coactivator 1; MEHP, mono(ethylhexyl) phthalate. 0026-895X/09/7505-1005–1013 MOLECULAR PHARMACOLOGY Vol. 75, No. 5 U.S. Government work not protected by U.S. copyright 53702/3462216 Mol Pharmacol 75:1005–1013, 2009 Printed in U.S.A. 1005 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from Results from these and other studies demonstrated that both CAR2 and CAR3 activate reporters containing response elements derived from the endogenous promoters of the CYP2B6 and CYP3A4 genes (Auerbach et al., 2005, 2007). Unlike CAR1, CAR3 functions as a ligand-dependent receptor (Auerbach et al., 2005), activating transcription in the presence of the human CAR ligand 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO) (Maglich et al., 2003). Our recent study of CAR2 suggested that it may have a ligand binding profile that is distinct from both CAR1 and CAR3 (Auerbach et al., 2007). Based on these data and results from molecular modeling analyses, we hypothesized that CAR2 possesses distinct biological properties. In this report, we demonstrate that CAR2 is a ligand-activated receptor that comprises approximately one third of the total CAR transcriptome in human hepatocytes. Furthermore, we identify the common plasticizer, di(2ethylhexyl) phthalate (DEHP), as a highly potent and uniquely selective agonist of CAR2. Results from reporter transactivation and mammalian two-hybrid assays reveal that DEHP activates CAR2 at low nanomolar concentrations. These results are further supported by analysis of CAR target gene expression in primary human hepatocytes, where DEHP activation of CAR2 markedly induced the CYP2B6 and CYP3A4 transcripts. Furthermore, using genomic analyses, we demonstrate that the alternative splice site used to generate the CAR2 transcript is not conserved in mice, rats, or marmosets—widely used animal models of DEHP toxicity. DEHP is a plasticizer with an annual production volume approaching 260 million pounds (Kavlock et al., 2006). Human exposure to DEHP is ubiquitous: it is used in the manufacturing of flexible vinyl found in medical tubing, other medical devices, commercial floorings, food product wraps, and many additional consumer products (Hauser and Calafat, 2005). Concerns regarding the human toxicology of DEHP are prevalent; for example, during medical interventions, patients can be exposed to relatively high doses of DEHP that leaches from plastic medical devices into infusate (United States Food and Drug Administration, 2001). Our finding that DEHP is a potent and selective activator of CAR2 is likely to have important implications for predictive human toxicity. In summary, in this report, we describe how the naturally occurring NR1I3 splice variant, CAR2, is unique in that it 1) is ligand activated, unlike the previously recognized constitutively active reference form of the receptor, 2) comprises 30% of the reference transcript level in human liver tissues, and, 3) is selectively activated by low nanomolar concentrations of DEHP, levels achieved in human serum and urine of certain patients. Furthermore, these studies yield new mechanistic insight for the toxicological effects of DEHP in human liver, findings that are particularly relevant because the conventional rodent and more recent marmoset models for DEHP toxicity do not reflect that of the human DEHP receptor, CAR2. Materials and Methods Chemicals. DEHP, 5 -androstan-3 -ol (androstanol), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). CITCO was obtained from BIOMOL Research Laboratories (Plymouth Meet-