CAR and PXR: the xenobiotic-sensing receptors

The xenobiotic receptors CAR and PXR constitute two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. In contrast, the steroid receptors, exemplified by the estrogen receptor (ER) and glucocorticoid receptor (GR), are the sensors that tightly monitor and respond to changes in circulating steroid hormone levels to maintain body homeostasis. This divergence of the chemical- and steroid-sensing functions has evolved to ensure the fidelity of the steroid hormone endocrine regulation while allowing development of metabolic elimination pathways for xenobiotics. The development of the xenobiotic receptors CAR and PXR also reflect the increasing complexity of metabolism in higher organisms, which necessitate novel mechanisms for handling and eliminating metabolic by-products and foreign compounds from the body. The purpose of this review is to discuss similarities and differences between the xenobiotic receptors CAR and PXR with the prototypical steroid hormone receptors ER and GR. Interesting differences in structure explain in part the divergence in function and activation mechanisms of CAR/PXR from ER/GR. In addition, the physiological roles of CAR and PXR will be reviewed, with discussion of interactions of CAR and PXR with endocrine signaling pathways.     

Androgen and Progesterone Receptors Are Targets for Bisphenol A (BPA), 4-Methyl-2,4-bis-(P-Hydroxyphenyl)Pent-1-Ene—A Potent Metabolite of BPA,and 4-Tert-Octylphenol: A Computational Insight

Exposure to toxic industrial chemicals that have capacity to disrupt the endocrine system, also known as endocrine disrupting chemicals (EDCs), has been increasingly associated with reproductive problems in human population. Bisphenol A (BPA; 4,4''-(propane-2,2-diyl)diphenol) and 4-tert-octylphenol (OP; 4-(1,1,3,3-tetramethylbutyl)phenol) are among the most common environmental contaminants possessing endocrine disruption properties and are present in plastics, epoxy resins, detergents and other commercial products of common personal and industrial use. A metabolite of BPA, 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is about 1000 times more biologically active compared to BPA. Epidemiological, clinical, and experimental studies have shown association of BPA and OP with adverse effects on male and female reproductive system in human and animals. The endocrine disruption activity can occur through multiple pathways including binding to steroid receptors. Androgen receptor (AR) and progesterone receptor (PR) are critical for reproductive tract growth and function. Structural binding characterization of BPA, MBP, and OP with AR and PR using molecular docking simulation approaches revealed novel interactions of BPA with PR, and MBP and OP with AR and PR. For BPA, MBP, and OP, five AR interacting residues Leu-701, Leu-704, Asn-705, Met-742, and Phe-764 overlapped with those of native AR ligand testosterone, and four PR interacting residues Leu-715, Leu-718, Met-756, and Met-759 overlapped with those of PR co-complex ligand, norethindrone. For both the receptors the binding strength of MBP was maximum among the three compounds. Thus, these compounds have the potential to block or interfere in the binding of the endogenous native AR and PR ligands and, hence, resulting in dysfunction. The knowledge of the key interactions and the important amino-acid residues also allows better prediction of potential of xenobiotic molecules for disrupting AR- and PR-mediated pathways, thus, helping in design of less potent alternatives for commercial use.     

The xenobiotic receptors PXR and CAR in liver physiology,an update

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two nuclear receptors that are well-known for their roles in xenobiotic detoxification by regulating the expression of drug-metabolizing enzymes and transporters. In addition to metabolizing drugs and other xenobiotics, the same enzymes and transporters are also responsible for the production and elimination of numerous endogenous chemicals, or endobiotics. Moreover, both PXR and CAR are highly expressed in the liver. As such, it is conceivable that PXR and CAR have major potentials to affect the pathophysiology of the liver by regulating the homeostasis of endobiotics. In recent years, the physiological functions of PXR and CAR in the liver have been extensively studied. Emerging evidence has suggested the roles of PXR and CAR in energy metabolism, bile acid homeostasis, cell proliferation, to name a few. This review summarizes the recent progress in our understanding of the roles of PXR and CAR in liver physiology.     

Monitoring Endocrine Disrupter Compounds in the Tunisian Hamdoun River using In Vitro Bioassays

Anthropogenic chemicals occurring in the environment, namely endocrine-disrupting chemicals (EDCs), have generated growing concern over their potential adverse effects on human wildlife health and ecosystem processes. This interest resulted particularly from their abilities to mimic the effect of endogenous hormones. In this study, we used stable transfected reporter cell lines to investigate the endocrine-disrupting profile of water as well as sediment samples. Samples are collected from up- and downstream of an industrial wastewater discharge point at the Hamdoun River in the vicinity of an industrial zone located at the center of Tunisia. The analysis of estrogen, androgen, and xenobiotic (pregnane X and dioxin) ligands receptors expressed by chimeric cell lines indicated that while the water and sediment samples from upstream sites have lower levels of estrogenic activity, those from downstream exhibited stronger estrogenic, aryl hydrocarbon receptor (AhR), and Pregnane X Receptor (PXR) activities. Moreover, collected samples have shown hormonal activity in terms of all tested receptors except the androgenic ones. In vitro recombinant estrogen receptor competitive binding assays revealed that while the estrogenic activities of the downstream water sample compounds had a strong affinity for estrogen receptor α (ERα), those present in the sediment samples showed a weaker one. These findings were consolidated by subsequent chemical analysis (high-performance liquid chromatography with UV detectors). Our results indicate that the water and sediment discharges at the Hamdoun River represent a major sink for EDCs from natural and industrial effluents, particularly those of the textile industry, with pernicious potential to disrupt normal endocrine functions.     

The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity

A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.     

Into the world of steroids: A biochemical “keep in touch” in plants and animals

Evolution of steroids such as sex hormones and ecdysteroids occurred independently in the animal and plant kingdoms. Plants use phytoecdysteroids (PEs) to control defense interactions with some predators; furthermore, PEs can exert beneficial influence on many aspects of mammalian metabolism. Endocrine disrupting compounds such as the estrogen agonist bisphenol A (BPA) are widespread in the environment, posing a potential hormonal risk to animals and plants. Adverse BPA effects on reproductive development and function are coupled with other toxic effects. BPA bioremediation techniques could be developed by exploiting some tolerant plant species.Key words: androgens, endocrine disrupting compounds, bisphenol A, estrogens, hormone receptors, phytoecdysteroids     

Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes

The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.     

Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution.     

Endocrine disrupting chemicals and disease susceptibility

Environmental chemicals have significant impacts on biological systems. Chemical exposures during early stages of development can disrupt normal patterns of development and thus dramatically alter disease susceptibility later in life. Endocrine disrupting chemicals (EDCs) interfere with the body's endocrine system and produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects in humans. A wide range of substances, both natural and man-made, are thought to cause endocrine disruption, including pharmaceuticals, dioxin and dioxin-like compounds, polychlorinated biphenyls, DDT and other pesticides, and components of plastics such as bisphenol A (BPA) and phthalates. EDCs are found in many everyday products--including plastic bottles, metal food cans, detergents, flame retardants, food additives, toys, cosmetics, and pesticides. EDCs interfere with the synthesis, secretion, transport, activity, or elimination of natural hormones. This interference can block or mimic hormone action, causing a wide range of effects. This review focuses on the mechanisms and modes of action by which EDCs alter hormone signaling. It also includes brief overviews of select disease endpoints associated with endocrine disruption.     

The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals

BACKGROUND: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. RESULTS: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. CONCLUSION: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.     

Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis

The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.     

Toxizität der Dioxine. Ultragifte oder Panikmache?

The toxicity of dioxins Dioxins are chloro‐organic compounds which show a high persistence in the environment. They are found, e.g., in food, blood and mother's milk. At certain doses, dioxins lead to pertubations of the immune and endocrine functions, developmental defects and tumors in experimental animals. In humans, a persistent form of acne, the so‐called chloracne, has been described. The toxicity of dioxins seems to depend on the activation of the dioxin‐ or arylhydrocarbon receptor. The physiological role of the receptor has not been elucidated so far.     

Nuclear receptors orchestrate detoxification pathways

Two nuclear receptors of xenobiotic drugs, PXR and CAR, are central regulators of detoxification enzymes. New studies extend the role of these receptors to a natural detoxification process. They coordinate induction of proteins for storage, glucuronidation, and canalicular transport of bilirubin.     

Functionally conserved xenobiotic responsive enhancer in cytochrome P450 3A7

Nuclear receptors CAR and PXR play a key role in cytochrome P450 gene induction by xenobiotics. Human cytochrome P450 3A7 (CYP3A7) is expressed from early in gestation until the perinatal period, when there is a switch in expression to CYP3A4. Here we demonstrate that a PXR and CAR responsive enhancer is located approximately 8 kb upstream of the proximal CYP3A7 promoter. This distal xenobiotic responsive enhancer module (XREM) is conserved with the XREM of CYP3A4. Interestingly, not only the XREM, but also the entire promoters exhibit 90% sequence identity up to -8.8 kb, indicating a close evolutionary distance. We propose that the promoters have coevolved to functionally conserve P450 gene induction in response to xenobiotics through CAR and PXR. Thus, nuclear receptors for xenobiotics may not only play a role to provide a survival advantage during adulthood, but also to protect the embryo against endogenous and exogenous toxins.