title: Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor.
abstract: Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant.
authors.affiliation: Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: DeKeyser
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Laurenzana
authors.forename: Eric C
authors.initials: EC
authors.lastname: Peterson
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.forename: Curtis J
authors.initials: CJ
authors.lastname: Omiecinski
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D015153:Blotting, Western; D019556:COS Cells; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis; D004051:Diethylhexyl Phthalate / Q000737:chemistry / Q000633:toxicity*; D052244:Endocrine Disruptors / Q000737:chemistry / Q000633:toxicity*; D004790:Enzyme Induction; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis; D000077297:Pregnane X Receptor; D011485:Protein Binding; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011987:Receptors, Steroid / Q000235:genetics*; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D000090702:Constitutive Androstane Receptor; D052244:Endocrine Disruptors; D008024:Ligands; D000077297:Pregnane X Receptor; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D004051:Diethylhexyl Phthalate; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating