BBA - General Subjects (v.1619, #3)

Cellular biology of cytochrome P450 regulation by Reinhard Lange (221-222).

Cytochrome P450s (P450s) constitute a superfamily of heme-proteins that play an important role in the activation of chemical carcinogens, detoxification of numerous xenobiotics as well as in the oxidative metabolism of endogenous compounds such as steroids, fatty acids, prostaglandins, and leukotrienes . In addition, some P450s have important roles in physiological processes, such as steroidogenesis and the maintenance of bile acid and cholesterol homeostasis . Given their importance, the molecular mechanisms of P450 gene regulation have been intensely studied. Direct interactions between transcription factors, including nuclear receptors, with the promoters of P450 genes represent one of the primary means by which the expression of these genes is controlled. In this review, several liver-enriched transcription factors that play a role in the tissue-specific, developmental, and temporal regulation of P450s are discussed. In addition, the nuclear receptors that play a role in the fine control of cholesterol and bile acid homeostasis, in part, through their modulation of specific P450s, are discussed.
Keywords: P450; Nuclear receptor; Liver-enriched transcription factor; Gene regulation; Knockout mouse; Transgenic mouse;

Functional and structural comparison of PXR and CAR by John T. Moore; Linda B. Moore; Jodi M. Maglich; Steve A. Kliewer (235-238).
The nuclear receptors pregnane X receptor (PXR, NR1I2) and constitutive active receptor (CAR, NR1I3) have both been proposed to function as xenosensors, but the details of their respective physiological roles are still being elucidated. We have contrasted these two receptors in a variety of experiments including gene expression assays, cell-based ligand profiling assays, and crystallographic/structural modeling analyses. These data highlight key differences between PXR and CAR.
Keywords: Nuclear receptor; Pregnane X receptor (PXR); Constitutive active receptor (CAR); Xenobiotic metabolism;

Phenobarbital induction of drug/steroid-metabolizing enzymes and nuclear receptor CAR by Satoru Kakizaki; Yukio Yamamoto; Akiko Ueda; Rick Moore; Tatsuya Sueyoshi; Masahiko Negishi (239-242).
Phenobarbital (PB) increases hepatic drug/steroid-metabolic capability by coordinately activating transcription of the genes encoding various metabolizing enzymes. The nuclear receptor CAR was first implicated as a transcription factor that activates the cytochrome P450 Cyp2b10 gene. In response to PB, CAR forms a heterodimer with the retinoid X receptor (RXR), binds to a PB response element (typified by DR-4 motif), and activates transcription of the gene. In the CAR-null mouse, PB does not only induce the Cyp2b10 gene, but also induces genes encoding various metabolizing enzymes. Thus, CAR is a general nuclear receptor that is essential for PB induction of drug/steroid metabolizing enzymes. PB also induces amino levulinate synthase 1 (ALAS-1), the rate-limiting enzyme in heme biosynthesis, to increase heme supply. However, PB induction of the synthase occurs in CAR-null mice, suggesting that CAR does not coordinate the heme synthesis for the induction of drug/steroid metabolism.
Keywords: Metabolizing enzyme; Nuclear receptor; Phenobarbital;

The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors by J.M. Pascussi; S. Gerbal-Chaloin; L. Drocourt; P. Maurel; M.J. Vilarem (243-253).
Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cytochromes P450 (CYPs), transferases and transporters. For example, natural and synthetic glucocorticoids (agonists and antagonists) as well as other clinically important drugs induce the hepatic CYP2B, CYP2C and CYP3A subfamilies in man, and these inductions might lead to clinically important drug–drug interactions. Only recently, the key cellular receptors that mediate such inductions have been identified. They include nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I2), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1). There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system appears now as a tangle of networks, where receptors share partners, ligands, DNA response elements and target genes. Moreover, they influence mutually their relative expression. This review is focused on these different pathways controlling human CYP2B6, CYP2C9 and CYP3A4 gene expression, and the cross-talk between these pathways.
Keywords: Nuclear receptor; Cytochrome P450; Gene expression regulation; Xenobiotic;

Phenobarbital (PB) responsiveness of CYP2B genes has been shown to be mediated by a PB responsive unit (PBRU). The core of the PBRU contains two nuclear receptor sites, NR-1 and NR-2, and a nuclear factor-1 (NF 1) binding site, which are required for PB responsiveness, but the importance of sequences flanking the core is not clear. We have used intravenous administration of plasmid DNA in the tail veins of mice to transfect hepatocytes in vivo and analyze sequence requirements for PB induction. In this assay PB treatment increased transactivation by the Cyp2b10 PBRU about 100-fold, which is similar to the increase in the expression of the endogenous gene while the Cyp2b9 PBRU was unresponsive. Analysis of chimeras of the two PBRUs and deletion mutants of the Cyp2b10 PBRU indicated that the core region containing the NR-1, NR-2 and NF-1 core sites is not sufficient for PB responsiveness. Additional sequence at the 3′ side of the core sequence, which included a previously defined accessory factor-1 (AF-1) site, partially restored responsiveness. This region contained a binding site for NF-1 only in Cyp2b10 and not in Cyp2b9, but the intact site was not required for PB responsiveness. Purified constitutive androstane receptor (CAR)/retinoid X receptor (RXR) bound to the core NR-1 and NR-2 sites and to a third NR-3 site to the 5′ side of the core in Cyp2b10. No binding of CAR/RXR to the Cyp2b9 PBRU was observed. These results indicate that changes in the NR sites which eliminate CAR/RXR binding are sufficient for the non-responsiveness to PB of Cyp2b9, but changes in sequences flanking the core independently eliminate PB responsiveness. The results demonstrate the advantages of transfection of mouse hepatocytes in vivo by tail vein injection of DNA as a method for transcriptional analysis of genes in vivo and show that sequences flanking the core region of the PBRU are required for PB induction in vivo.
Keywords: Cytochrome P450; In vivo gene delivery; Phenobarbital; Cyp2b gene; Nuclear receptor;

Functional role of AhR in the expression of toxic effects by TCDD by Junsei Mimura; Yoshiaki Fujii-Kuriyama (263-268).
Cytochrome P450 1A1 (CYP1A1) is one of the xenobiotic metabolizing enzymes (XMEs), which is induced by polycyclic aromatic hydrocarbons (PAHs). The most potent inducer of CYP1A1 is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, TCDD induces a broad spectrum of biochemical and toxic effects, such as teratogenesis, immunosuppression and tumor promotion. Most, if not all, of the effects caused by TCDD and other PAHs are known to be mediated by AhR (aryl hydrocarbon receptor or dioxin receptor) which has a high binding affinity to TCDD. The liganded AhR translocates from cytoplasm to nuclei where it switches its partner molecule from Hsp90 to Arnt. Thus formed AhR/Arnt heterodimer binds a specific DNA sequence designated XRE in the promoter region of the target genes including CYP1A1, UDP-glucuronosyl transferase and others to enhance their expression. Although it remains to be studied how AhR is involved in the other TCDD-induced biological effects such as teratogenesis and immunosuppression than induction of XMEs, it is believed that these adverse TCDD effects are caused by untimely activation of gene expression by ligand-activated AhR in the biological process. We summarize the present knowledge about functional role of AhR in TCDD-induced biological effects.
Keywords: Arylhydrocarbon receptor; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; CYP1A1;

Transcriptional response elements in the promoter of CYP6B1, an insect P450 gene regulated by plant chemicals by Rebecca A. Petersen; Hataichanoke Niamsup; May R. Berenbaum; Mary A. Schuler (269-282).
Papilio polyxenes, a lepidopteran continually exposed to toxic furanocoumarins in its hostplants, owes its tolerance to these compounds to the transcriptional induction of the CYP6B1 gene encoding a P450 capable of metabolizing linear furanocoumarins, such as xanthotoxin, at high rates. Transient expression of various lengths of wild-type and mutant CYP6B1v3 promoter in lepidopteran Sf9 cells defines a positive element (XRE-xan) from −136 to −119 required for both basal and xanthotoxin-inducible transcription and a negative element from −228 to −146 that represses basal transcription. Fusion of the CYP6B1v3 XRE-xan element to the Drosophila melanogaster Eip28/29 core promoter indicates that the XRE-xan functions in conjunction with its own core promoter but not with a heterologous core promoter. Sequence searches of the CYP6B1v3 proximal promoter region revealed a number of putative elements (XRE-AhR, ARE, OCT-1, EcRE, C/EBP, Inr) sharing sequence similarity with those in other regulated vertebrate and insect promoters. Mutation of TGAC nucleotides shared by the overlapping EcRE/ARE/XRE-xan indicates that this sequence is essential for basal and regulated transcription of this gene. Mutagenesis in the non-overlapping region of the EcRE indicates it modulates basal transcription. These findings are incorporated into a working model for regulation of this toxin-inducible promoter.
Keywords: Papilio polyxenes; Cytochrome P450 monooxygenase; Furanocoumarin; XRE-xan;

Alcohol and tobacco are frequently co-abused. Increased alcohol use and alcoholism are associated with smoking, and vice versa. Functional and/or metabolic cross-tolerance may contribute to this occurrence. This review summarizes recent studies published from our laboratory focusing on metabolic aspects of tolerance, which demonstrate that in rat, subchronic, behaviourally relevant doses of ethanol induce hepatic nicotine-metabolizing cytochrome P450 (CYP) 2B1, and that subchronically administered nicotine, at behaviourally relevant doses, induces hepatic ethanol-metabolizing CYP2E1. Increased CYP2B1 protein, mRNA and CYP2B1-mediated nicotine metabolism was observed following ethanol treatments. CYP2E1 protein and activity were induced by nicotine, but no changes were seen in levels of CYP2E1 mRNA. These data indicate that metabolic cross-tolerance may occur between nicotine and ethanol, suggesting that nicotine use may increase the elimination of ethanol, and ethanol use may increase the elimination of nicotine. Other implications, such as altered pharmacology and toxicology of drugs metabolized by these enzymes, as well as changes in pro-carcinogen and pro-toxin activation are also discussed.
Keywords: Cytochrome P450; Nicotine; Ethanol; Cross-tolerance; Rat; Drug metabolism; Hepatotoxicity;

The cytochrome P450c17 isoforms from various animal species have different substrate selectivity, especially for 17,20-lyase activity. In particular, the human P450c17 selectively produces dehydroepiandrosterone with little androstenedione (AD). Hamster P450c17, on the other hand, produces both of these steroids at comparable rates. We thus investigated if computational analysis could explain the difference in activity profiles. Therefore, we inserted the four P450c17 substrates—pregnenolone, progesterone, and their 17α-hydroxylated forms—inside our hamster P450c17 model, which we derived from our human P450c17 model based on the crystal structure of P450BMP. We performed molecular dynamics (MD) simulations on the complexes and analyzed the resultant trajectories to identify amino acids that interact with substrates. Starting with substrates in two different orientations, we obtained two sets of binding trajectories in each case. The first set of trajectories reveal structural rearrangements that occur during binding, whereas the second set of trajectories reflects substrate orientations during catalysis. Our modeling suggests that three distinct steps are required for substrate selectivity and binding to the hamster P450c17: (1) recognition of the substrate at the putative substrate entrance, characterized by a pocket at the surface of the hamster P450c17 containing charged residues R96 and D116; (2) entry of the substrate into the active site, in an intermediate position directed by possible hydrogen bonding of the substrates with the heme D-ring propionate group, R96, R440, and T306; followed by (3) 90° counterclockwise rotation of the substrates, positioning them in optimal position for reactivity, a process that may be directed by hydrogen bonding to the 110–112 region of the hamster P450c17. With some substrates, we obtained trajectories which suggest that major distortions in the I-helix and opening of the H–I loop occur during substrate binding. In conclusion, these modeling exercises provide insight to possible structural reorganizations that occur during substrate binding and suggest that amino acids that participate in three distinct steps of this process may all contribute to substrate binding and activity.
Keywords: Cytochrome P450c17 (CYP17); 17α-Hydroxylase/17,20-lyase; Hamster adrenal gland; Computer modeling; Molecular dynamics; Steroid hormone;

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction by Keisuke Shibuya; Norio Takata; Yasushi Hojo; Aizo Furukawa; Nobuaki Yasumatsu; Tetsuya Kimoto; Taihei Enami; Kumiko Suzuki; Nobuaki Tanabe; Hirotaka Ishii; Hideo Mukai; Taiki Takahashi; Taka-aki Hattori; Suguru Kawato (301-316).
Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17β-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017α and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca2+ influx attendant upon neuron–neuron communication via N-methyl-d-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron–neuron communication, thereby mediating learning and memory in the hippocampus.
Keywords: neurosteroid; P450; hippocampus; brain; LTP; signal transduction;

The undifferentiated cell zone is a stem cell zone in adult rat adrenal cortex by Fumiko Mitani; Kuniaki Mukai; Hirokuni Miyamoto; Makoto Suematsu; Yuzuru Ishimura (317-324).
The adrenal cortex of mammals has been known to consist of three morphologically and functionally distinct zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF) and the zona reticularis (zR), each of which secretes a specific corticosteroid different from those produced by the other two zones. We found previously, however, that an additional zone existed between zG and zF of adult rat adrenal cortex and that the cells in that zone were in a functionally undifferentiated state as an adrenocortical cell [Endocrinology 135, (1994) 431]: they were incapable of synthesizing highly active forms of corticosteroids, such as aldosterone and corticosterone, although they could produce their precursors. Hence, we named the zone as the undifferentiated cell zone (zU) of the adrenal cortex. Here we show that zU and its surroundings, i.e. the innermost portion of zG and the outermost portion of zF are the sites for cell replication in adult rat adrenal cortex and that the cells raised there migrate to other regions. Such cell replications in this region occur regardless of physiological conditions, such as the rise and fall of hormonal stimuli and circadian fluctuation of adrenocortical activities. On the bases of these and other findings previously described, we propose that zU is the stem cell zone of the adult rat adrenal cortex. Our recent success in isolating novel cell lines, which display an undifferentiated phenotype similar to that of zU cells, could facilitate the exploration of molecular mechanisms for the differentiation and development of the adrenocortical cells.
Keywords: Stem cell; Adrenal cortex; Circadian rhythm; Cell differentiation; Steroidogenesis; Cytogenesis;

Expression of cytochromes P450 4F4 and 4F5 in infection and injury models of inflammation by X Cui; A Kalsotra; A.M Robida; D Matzilevich; A.N Moore; C.L Boehme; E.T Morgan; P.K Dash; H.W Strobel (325-331).
Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.
Keywords: Regulation of CYP 4F expression; Inflammation and P450 expression; Brain cytochrome P450 distribution; Lipopolysaccharide and CYP 4F subfamily expression; CYP 4F subfamily expression; CYP 4F subfamily suppression by inflammation;

Cytochrome P450 epoxygenases and vascular tone: novel role for HMG-CoA reductase inhibitors in the regulation of CYP 2C expression by Beate Fisslthaler; U.Ruth Michaelis; Voahanginirina Randriamboavonjy; Rudi Busse; Ingrid Fleming (332-339).
Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI2)-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI2-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.
Keywords: Cytochrome P450 2C; Endothelial cell; Endothelium-derived hyperpolarizing factor; Epoxyeicosatrienoic acid; Nitrotyrosine; Reactive oxygen species;