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Abstract: Caco-2 cell line is extensively used as an in vitro model in studying small intestinal absorption but it lacks proper expression of efflux pumps and cytochrome P450 enzymes that are involved in absorption and first pass metabolism of drugs. We created two novel Caco-2 cell lines expressing orphan nuclear receptors pregnane X receptor and constitutive androstane receptor that regulate many genes involved in xenobiotic metabolism. We conducted a systematic study on expression of some metabolic genes, P-glycoprotein activity and absorption properties of several drugs with these new cell lines and previously described modified Caco-2 cell lines (MDR1 transfection, vincristine treatment and 1α,25-dihydroxyvitamin D3 treatment). A short culture time medium was also included in the study. Most modified cell lines formed tight differentiated monolayers. MDR1, CYP2C9 and CYP3A4 genes were upregulated in some cell lines. Elevated P-glycoprotein activities were observed by calcein-AM uptake experiments but this did not affect significantly the permeability of selected P-glycoprotein substrates. Some cell lines had similar passive and active permeability properties to Caco/WT cells while in few cell lines these were altered. Passive transcellular permeability was modestly elevated in all modified cell lines. In addition, several compounds showed pH-dependent permeability properties. [Copyright &y& Elsevier]

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells. These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1). MDCKII cells that overexpress these proteins (MDCKII–MDR1 or MDCKII–MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay. Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay.Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability. This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity. The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII–MDR1 cell line.This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay. Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial. Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies. [Copyright &y& Elsevier]

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-α, 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. [Copyright &y& Elsevier]

Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with carrier DNA complexes have been studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were tested.Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. Furthermore, preliminary results suggest that cell surface GAGs, particularly heparan sulfate, inhibit gene transfer by cationic lipids and polymers. [Copyright &y& Elsevier]

Abstract: WERI-Rb1 retinoblastoma (Rb) cell line, a human photoreceptor model, is notoriously difficult to transfect. Culturing of the WERI-Rb1 cells as a monolayer is complicated and cells are easily detached during transfection. Furthermore, transfection efficiencies in monolayer and in suspension are moderate at best which has limited the analysis of photoreceptor-specific promoters with low activity. To overcome these limitations, we developed a straightforward reverse transfection method for WERI-Rb1 cells wherein snap-frozen DNA/polyethylenimine complexes are freeze-dried on the surface of 48-well plates and stored in desiccator until cells are seeded for transfection. Comparing to conventional transfection, reverse transfection turned out to have equal or better transfection efficiency. In addition, while conventional transfection with cationic polymers requires serum-free conditions, reverse transfection can be performed in the presence of serum. Importantly, DNA/polyethylenimine complexes promote cell adhesion to the plates. This enables cell culturing as monolayers with concurrent complex uptake. Also, long-term storage of the plates did not reduce the transfection efficiency nor it had any effects on the cell toxicity. Because of the stability of complexes, reverse transfection enables large-scale transfection of hard-to-transfect retinoblastoma cells thus providing a reproducible, cost-effective and versatile tool for parallel screening of proteins and gene regulatory elements used in diverse applications. [Copyright &y& Elsevier]

We have studied retina-specific gene expression and gene promoter activity in WERI-Rb1 retinoblastoma cells. In general, the expression of endogenous genes matched the efficiency of promoter activity of the transfected gene: interphotoreceptor retinoid binding protein and phosphodiesterase-β mRNAs and reporter activities were readily detected while other retina-specific messages were at or below the detection limit in WERI-Rb1 cells. Phosphodiesterase-β promoter appeared active in all six cell lines tested. The viral SV40 promoter is very weak in WERI-Rb1 cells, which has implications for its use in gene constructs targeted to the photoreceptors. Our results also show that polyethyleneimine 25 is an efficient and simple carrier for DNA. The optimized transfection conditions permit the use of 24-well plates and low amounts of DNA for improved analysis of promoter activities, as compared to previous studies. Our results are expected to facilitate further research on retina-specific gene expression. [Copyright &y& Elsevier]

Abstract Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g. , bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [3H]taurocholate (TCA), [3H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTβ and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. [ABSTRACT FROM AUTHOR]

The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells. [ABSTRACT FROM AUTHOR]

Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z ′ factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] FL442 is selective to AR over other closely related steroid hormone receptors. [•] FL442 exhibits equal efficacy to prevent PCa cell proliferation as enzalutamide. [•] FL442 maintains antiandrogenic activity with enzalutamide-activated ARF876L. [•] FL442 accumulates strongly in the mouse prostate. [•] FL442 significantly inhibits LNCaP xenograft tumor growth. [Copyright &y& Elsevier]

[Display omitted] Quantitation of ocular drug metabolism is important, but only sparse data is currently available. Herein, the pharmacokinetics of four drugs, substrates of metabolizing enzymes, was investigated in albino rabbit eyes after intracameral and intravitreal administrations. Acetaminophen, brimonidine, cefuroxime axetil, and sunitinib and their corresponding metabolites were quantitated in the cornea, iris-ciliary body, aqueous humor, lens, vitreous humor, and neural retina with LC-MS/MS analytics. Non-compartmental analysis was employed to estimate the pharmacokinetic parameters of the parent drugs and metabolites. The area under the curve (AUC) values of metabolites were 12–70 times lower than the AUC values of the parent drugs in the tissues with the highest enzymatic activity. The ester prodrug cefuroxime axetil was an exception because it was efficiently and quantitatively converted to cefuroxime in the ocular tissues. In contrast to the liver, sulfotransferases, aldehyde oxidase, and cytochrome P450 3A activities were low in the eye and they had negligible impact on ocular drug clearance. With the exception of esterase substrates, metabolism seems to be a minor player in ocular pharmacokinetics. However, metabolites might contribute to ocular toxicity, and drug metabolism in various eye tissues should be investigated and understood thoroughly. [ABSTRACT FROM AUTHOR]

Highlights: [•] Misclassification of Curcuma elata leads to unwanted human exposure to sesquiterpenes. [•] Sesquiterpenes activate human PXR and CAR, and consequently, CYP expression. [•] Reactive sesquiterpene metabolites are formed by human liver microsomes. [•] CYP-mediated metabolism is the probable reason for observed liver toxicity. [•] Due to CYP inhibition by sesquiterpenes, herb–drug interactions are also likely to occur. [ABSTRACT FROM AUTHOR]

Abstract: A three-dimensional micro-scale perfusion-based two-chamber (3D-μPTC) tissue model system was developed to test the cytotoxicity of anticancer drugs in conjunction with liver metabolism. Liver cells with different cytochrome P450 (CYP) subtypes and glioblastoma multiforme (GBM) brain cancer cells were cultured in two separate chambers connected in tandem. Both chambers contained a 3D tissue engineering scaffold fabricated with biodegradable poly(lactic acid) (PLA) using a solvent-free approach. We used this model system to test the cytotoxicity of anticancer drugs, including temozolomide (TMZ) and ifosfamide (IFO). With the liver cells, TMZ showed a much lower toxicity to GBM cells under both 2D and 3D cell culture conditions. Comparing 2D, GBM cells cultured in 3D had much high viability under TMZ treatment. IFO was used to test the CYP-related metabolic effects. Cells with different expression levels of CYP3A4 differed dramatically in their ability to activate IFO, which led to strong metabolism-dependent cytotoxicity to GBM cells. These results demonstrate that our 3D-μPTC system could provide a more physiologically realistic in vitro environment than the current 2D monolayers for testing metabolism-dependent toxicity of anticancer drugs. It could therefore be used as an important platform for better prediction of drug dosing and schedule towards personalized medicine. [Copyright &y& Elsevier]

Abstract: Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR. [Copyright &y& Elsevier]

Abstract: The so-called human xenosensors, constitutive androstane receptor (hCAR), pregnane X receptor (hPXR) and aryl hydrocarbon receptor (hAhR), participate in drug metabolism and transport as well as in several endogenous processes by regulating the expression of their target genes. While the ligand specificities for hPXR and hAhR are relatively well described, this property of hCAR still remains fairly unclear. Identifying hCAR agonists for drug development and for studying hCAR biology are hindered mainly by the unique properties of the receptor, such as the high constitutive activity and complex signaling network but also by the lack of robust and reliable assays and cellular models. Here, validated reporter assays for these three xenosensors are presented and thereafter used to screen a large set of chemicals in order to find novel selective hCAR ligands. We introduce a novel selective hCAR agonist, FL81, which can be used as a stable positive control in hCAR activity assays. Our established receptor-selective ligand identification methods consisting of supporting biological assays and molecular modeling techniques are then used to study FL81 as well as other discovered ligands, such as diethylstilbestrol, o,p′-DDT, methoxychlor and permethrin, for their ability to specifically activate hCAR and to regulate the CYP enzyme expression and function. [Copyright &y& Elsevier]

Abstract: In this work, 52 diphenyl-4,5-dihydroisoxazoles and -3-hydroxy ketones were prepared and their estrogen receptor α (ERα) and estrogen receptor β (ERβ) activities were explored in order to systematize and maximize their biological activity. The biological activity was firstly screened by using ERE reporter assay to find out how aromatic hydroxylation and methylation of the chiral centers of the compounds affect the ability of ER to mediate biological responses. For selected 19 compounds, the relative binding affinities (RBA, relative to 3,17β-estradiol) and ability to induce transcription of primary E2 target gene pS2 in human MCF-7 breast cancer cells were determined. In the reporter assay, many compounds showed even stronger activity than E2 and some of them showed RBA larger than 1%. The highest RBAs were determined for the enantiomers of 1-hydroxy-6-(4-hydroxy-phenyl)-1-phenyl-hexan-3-one (50a and 50b). Isomer 50a showed high binding affinity both to ERα (with RBA ∼200%) and ERβ (with RBA ∼60%), while the RBAs of 50b were ca. 40% of those. Some of the other compounds (with RBA ∼1–16%) showed also notable ERα binding selectivity. When four most promising ligands (50a, 50b, 45a, and 45b) were studied with respect to their ability to induce the transcription of primary E2 target gene pS2, the compounds acted as agonists or partial agonists. Computer modeling was used to predict receptor binding conformations and to rationalize the RBA differences of the compounds. [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1–6 (MRP1–6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087–1098, 2010 [ABSTRACT FROM AUTHOR]

Abstract: The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor responsible for the recognition of potentially toxic endo- and exogenous compounds whose elimination from the body is accelerated by the CAR-mediated inducible expression of metabolizing enzymes and transporters. Despite the importance of CAR, few human agonists are known so far. Following a sequential virtual screening procedure using a 3D pharmacophore and molecular docking approach, we identified 17 novel agonists that could activate human CAR in vitro and enhance its association with the nuclear receptor co-activator SRC1. Selected agonists also increased the expression of the human CAR target CYP2B6 mRNA in primary hepatocytes. Composed of substituted sulfonamides and thiazolidin-4-one derivatives, these agonists represent two novel chemotypes capable of human CAR activation, thus broadening the agonist spectrum of CAR. [Copyright &y& Elsevier]

Abstract: The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca2+, Ba2+, Sr2+ and combination of Ca2+ and Ba2+), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca2+ and Ba2+ ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca2+ and Ba2+ cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca2+ and Ba2+ have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50kDa; radius of gyration of 3nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca2+ and Ba2+. This combination may be useful in cell therapy. [Copyright &y& Elsevier]

Abstract: The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity. [Copyright &y& Elsevier]

The human constitutive androstane receptor (CAR, NR1I3) is an important ligand-activated regulator of oxidative and conjugative enzymes and transport proteins. Because of the lack of a crystal structure of the ligand-binding domain (LBD), wide species differences in ligand specificity and the scarcity of well characterized ligands, the factors that determine CAR ligand specificity are not clear. To address this issue, we developed highly defined homology models of human CAR LBD to identify residues lining the ligand-binding pocket and to perform molecular dynamics simulations with known human CAR modulators. The roles of 22 LBD residues for basal activity, ligand selectivity, and interactions with co-regulators were studied using site-directed mutagenesis, mammalian co-transfection, and yeast two-hybrid assays. These studies identified several amino acids within helices 3 (Asn165), 5 (Val199), 11 (Tyr326, Ile330, and Gln331), and 12 (Leu343 and Ile346) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile164 and Asn165), 5 (Cys202 and His203), and 7 (Phe234 and Phe238) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe243) appears to explain the human/mouse species difference in response of CAR to 17α-ethynyl-3,17β-estradiol. [ABSTRACT FROM AUTHOR]