Induction of cytochrome P4501A (CYP1A) by clotrimazole, a non-planar aromatic compound. Computational studies on structural features of clotrimazole and related imidazole derivatives (original) (raw)
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INDUCTION OF CYP1A BY THE N-IMIDAZOLE DERIVATIVE, 1-BENZYLIMIDAZOLE
Environmental Toxicology and Chemistry, 2003
Xenobiotics can induce cytochrome P4501A (CYP1A) by ligand binding to the aryl hydrocarbon receptor (AhR). Typical AhR ligands are polycyclic aromatic compounds with planar molecular conformation. The present work investigated the ability of the N-imidazole derivative, 1-benzylimidazole (BIM), to induce CYP1A in rainbow trout hepatocytes. Benzylimidazole increased hepatocellular CYP1A catalytic activity (determined as 7-ethoxyresorufin-O-deethylase [EROD] activity) and CYP1A mRNA in a concentration-dependent way. Computational studies on the molecular structure of BIM indicated that the energetically most stable BIM conformer has the imidazole ring and the phenyl ring in different planes, i.e., does not take a planar conformation. This property of BIM does not agree with the structural requirements of a typical AhR ligand. In line with this observation, we found that the AhR antagonist, ␣-naphthoflavone (␣NF), was not able to inhibit BIM induction of EROD activity and CYP1A mRNA, although it inhibited the induction of CYP1A by the prototypic AhR ligand, -naphthoflavone (NF). The results suggest that transcriptional activation of CYP1A by the N-imidazole derivative, BIM, is not mediated through direct ligand binding to the AhR.
Toxicology in Vitro, 2005
A variety of aquatic pollutants are able to induce cytochrome P4501A (CYP1A) in fish by ligand binding to the aryl hydrocarbon receptor (AhR). High-affinity AhR ligands are planar aromatic polycyclic molecules such as the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present work investigates the ability of the imidazole derivative, Prochloraz (PRO), to induce CYP1A. Computational studies on the molecular structure of PRO indicated that it is highly unlikely for PRO to have both aromatic rings of the molecule, i.e. the imidazole and the benzene ring, in the same plane. Thus, the possible conformers do not take planar structures, in contrast to the typically planar AhR ligands. Experimentally, the capability of PRO to induce CYP1A was assessed using the rainbow trout liver cell line, RTL-W1, as in vitro model. PRO increased in a concentration-dependent way the catalytic activity of CYP1A (determined as 7-ethoxyresorufin-O-deethylase, EROD, activity) in RTL-W1 cells. The potency of PRO was lower than that of a reference AhR-ligand, b-naphthoflavone (bNF). In addition to the catalytic level, PRO activated CYP1A also at the transcriptional level as determined by RT-PCR analysis of CYP1A mRNA. These results indicate that PRO, although its structure is not corresponding to the typical features of CYP1A-inducing AhR ligands, still is able to activate CYP1A expression.
Toxicology in Vitro, 2007
The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, b-naphthoflavone (bNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of bNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist a-naphthoflavone (aNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. aNF and herbimicin provoked a decrease of EROD induction both by bNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of bNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.
Archives of Biochemistry and Biophysics, 1996
The aryl hydrocarbon (Ah) 2 receptor is a cytoplasmic receptor, which is involved in CYP1A1 induction by Three benzimidazole compounds, omeprazole (OP), aryl hydrocarbons. The structural characteristics of the thiabendazole (TBZ), and lansoprazole (LP), were compared with respect to the induction of CYP1A1-mRNA Ah receptor are (1) a basic helix-loop-helix (bHLH) in human hepatoma cells, HepG2. OP was the most domain at the N-terminal, (2) PAS (Per, ARNT, and potent inducer among the three compounds, but LP Sim gene products have a high similar repeat of amino was found to be a weak inducer. Induction by TBZ acids) domains in the middle of the molecule, and (3) was at an intermediate level. None of these compounds an activation domain of transcription at the C-termiinduced CYP1A1-mRNA in a mouse hepatoma cell line, nal. The Ah receptor forms a complex with 90-kDa heat Hepa-1. The transient expression of mouse Cyp1a1shock protein (HSP90) and this complex is localized in CAT gene into HepG2 cells showed that OP treatment the cytoplasm. After ligand binding to the Ah receptor, of the transfectants induced CAT activity to the same this receptor may change its conformation, release degree as 2,3,7,8-tetrachlorodibenzo-p-dioxin treat-HSP90 from the complex, and form a heterodimer with ment. Therefore, the cellular factors in human cells the Ah receptor nuclear translocator (ARNT). This hetwere able to work on the mouse regulatory element. erodimer can bind to the xenobiotic response element The expression of human aryl hydrocarbon (Ah) recep-(XRE), which is a 5-upstream-regulatory element of tor in the mouse Hepa-1 mutant cell line cl-19, which the CYP1A1 gene, and the transcription complex can is defective in Ah receptor, did not increase the inducinitiate the transcription of the CYP1A1 gene (see retion level of CYP1A1-mRNA by OP treatment. When cent review articles (1, 2)). However, it has been rethe cultured medium of HepG2 cells in the presence of ported that some benzimidazole compounds, such as OP was added to the mouse Hepa-1 cell culture meomeprazole (3), lansoprazole (4), and thiabendazole (5), dium, CYP1A1-mRNA was not induced in Hepa-1 cells. are potent inducers for the CYP1A1 gene product, but It is thus concluded that metabolites of OP in human do not bind to the Ah receptor. cells are not the ligands for the human Ah receptor. In a previous report, we showed the human-specific Therefore, in human cells, but not mouse cells, there induction of CYP1A1 resulting from OP treatment, usmust be an OP-sensitive activation factor for the huing five cell lines (6). Taking advantage of the species man Ah receptor.
European Journal of Biochemistry, 1996
The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYPIAI gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer p-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYPlAl gene transcription, and to a superinduction when combined with either P-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer Pnaphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable doselresponse and rate of induction of CYPIAI gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to pnaphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYPlAl transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracel-M a r signaling, initiated by benzimidazole derivatives.
Aquatic Toxicology, 1993
The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine 'dioxin equivalents' in environmental samples, including extracts of fish tissues. However, the relative potency of inducing compounds may vary between species, suggesting the need for taxonspecific model systems. In this paper we present an initial characterization of CYP1A induction in one such system, a teleost liver cell line (PLHC-1) derived from a hepatocellular carcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988. J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligand 2-azido-3-[~zsI]iodo-7,8-dibromodibenzo-p-dioxin ([~25I]NaBr2DD) to proteins in PLHC-1 cytosol indicated the presence of the Ah receptor, which is known to control CYP1A induction in mammals. 3,3',4,4'-Tetrachlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells that was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYP1 A1 (P450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12 3 showed specific recognition of CYP1A induced by TCB. No staining was seen in untreated or vehicle-treated cells. There was an excellent quantitative correlation between amounts of CYPI A protein detected immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-deethylase (EROD) activity occurred at 0.1 ¢tM TCB; at higher concentrations (1 and 10/tM), EROD activity was reduced as compared to the activity at 0.1 ¢tM TCB. In contrast, immunoreactive CYP1A protein increased with increasing TCB concentration. up to 10/.tM. The loss of EROD activity at high concentrations of TCB did not result from changes in cell number or viability. The apparent inhibition or inactivation of CYP1A catalytic activity by the higher Abbreviations used: AHH, aryl hydrocarbon hydroxylase; BCIP, 5-bromo-4-chloro-3-indolylphosphate; BNF, fl-naphthoflavone; ECs0, estimated concentration needed to produce 50% of the maximal response; EROD, ethoxyresorufin O-deethylase; HAH, halogenated aromatic hydrocarbons; MAb, monoclonal antibody; [~25I]N3Br2DD, 2-azido-3-[125I]iodo-7,8-dibromodibenzo_p_dioxin; NBT, nitro blue tetrazolium; P450, cytochrome P450; PAH, polynuclear aromatic hydrocarbons; PBS, phosphate-buffered saline; PCB, polychlorinated biphenyl; TEF, toxic equivalency factor; TCB, 3,3',4, TCDD, 2,3,7, TCDF, 2,3,7, concentrations of halogenated biphenyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 ceils may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing potency of halogenated aromatic hydrocarbon congeners, and for investigating the mechanism of CYP1A inhibition or inactivation by environmental contaminants such as TCB.
Inorg Chem, 2022
The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe−N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe−N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.
Journal of Biological Chemistry, 2003
Cytochrome P-450 1A1 (CYP1A1) is known to be induced by aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), through activation of the aryl hydrocarbon receptor (AhR). We found that p38 MAP kinase inhibitors (SB203580 and SB202190; 40 M each; pyridinyl imidazole compounds) suppressed CYP1A1-mRNA induction by TCDD (2 nM) in mouse hepatoma Hepa-1 cells and in human hepatoma HepG2 cells, and also suppressed CYP1B1-mRNA induction by TCDD (2 nM) in human breast adenocarcinoma MCF7 cells. An analogue compound, SB202474, which does not inhibit p38 MAP kinase, also suppressed CYP1A1-mRNA induction by TCDD. Moreover, overexpression of a dominantnegative gene for p38 MAP kinase in Hepa-1 cells did not suppress Cyp1a1 reporter gene induction by TCDD. Therefore, the suppression of Cyp1a1 transcription by pyridinyl imidazole compounds is not because of their inhibition of p38 MAP kinase activity. Because SB203580 did not inhibit in vitro AhR transformation by TCDD, this compound was not acting as a simple AhR antagonist. SB203580 decreased TCDD-induced histone acetylation levels in the region of the Cyp1a1 gene promoter, especially around the TATA box sequence. This result suggests the possibility that pyridinyl imidazole compounds suppress the recruitment of some co-activator that has the histone acetyltransferase activity necessary for CYP1A1-mRNA transcription.
Biochemical pharmacology, 2016
6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching freq...