Báo cáo khoa học: Modulation of aryl hydrocarbon receptor transactivation by carbaryl, a nonconventional ligand pptx

Modulation of aryl hydrocarbon receptor transactivation by carbaryl, a nonconventional ligand ´ ˜ Susanna Boronat1, Susana Casado2, Jose M Navas2 and Benjamin Pina1 ´ Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Cientıficas, Barcelona, Spain ´ Department of Environment, Instituto Nacional de Investigacion y Tecnolog’a Agraria y Alimentaria (INIA), Madrid, Spain Keywords bioassays; dioxin-like; endocrine disruptors; recombinant yeast assays; transcriptional response Correspondence B Pina, IBMB-CSIC, Jordi Girona, ˜ 18, 08034 Barcelona, Spain Fax: +34 93 204 59 04 Tel: +34 93 400 61 57 E-mail: bpcbmc@cid.csic.es (Received March 2007, revised May 2007, accepted May 2007) doi:10.1111/j.1742-4658.2007.05867.x Carbaryl (1-naphthyl-N-methylcarbamate), a widely used carbamate insecticide, induces cytochrome P450 1A gene expression in mammalian cells This activity is usually mediated by the interaction of the compound with the aryl hydrocarbon receptor However, it has been proposed that this mechanism does not apply to carbaryl because its structure differs from that of typical aryl hydrocarbon receptor ligands We show here that carbaryl promotes activation of target genes in a yeast-based bioassay expressing both aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator By contrast, carbaryl acted as a competitive inhibitor, rather than as an agonist, in a simplified yeast system, in which aryl hydrocarbon receptor nuclear translocator function is bypassed by fusing aryl hydrocarbon receptor to a heterologous DNA binding domain This dual action of carbaryl, agonist and partial antagonist, was also observed by comparing carbaryl response in two vertebrate cell lines A yeast two-hybrid assay showed that the mammalian coactivator cAMP response element-binding protein readily interacts with aryl hydrocarbon receptor bound to its canonical ligand b-naphthoflavone, but not with the carbaryl–aryl hydrocarbon receptor complex We propose that carbaryl interacts with aryl hydrocarbon receptor, but that its peculiar structure imposes a substandard configuration on the aryl hydrocarbon receptor ligand-binding domain that prevents interaction with key coactivators and activates transcription without the need for aryl hydrocarbon receptor nuclear translocator This effect may be relevant in explaining its physiological effects in exposed animals, and may help to predict its effects, and that of similar compounds, in humans Our data also identify the aryl hydrocarbon receptor ⁄ cAMP response element-binding protein interaction as a molecular target for the identification and development of new aryl hydrocarbon receptor antagonists The known or suspected deleterious effects of global pollution by different chemical species, ranging from industrial by-products to pesticides, has developed into a major public concern in recent decades Each year, thousands of new chemicals are released into the environment at a pace that makes impossible the precise characterization of their acute and ⁄ or chronic impact, both on human health and on exposed ecosystems Abbreviations AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; BNF, b-naphthoflavone; CBP, cAMP response element-binding protein; DBD, DNA binding domain; GUS, b-glucuronidase; HAT, histone acetyltransferase; LBD, ligand-binding domain; LOEC, lowest observed effect concentration; RTL, rainbow trout liver; RYA, recombinant yeast assay; TCDD, 2,3,7,8-tetrachlorodibenzo(p)dioxin; XRE, xenobiotic responsive element FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3327 Modulation of AhR transactivation by carbaryl S Boronat et al Among the different types of pollutants, those interacting with cell receptors constitute an even greater risk as they become toxic at very low concentrations, sometimes at, or under, the limits of detection by conventional analytical procedures [1] The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix-PAS family of transcription regulators [2] This family roots itself on the prokaryotic kingdom; however, the capacity to bind specific ligands and to modulate the transcriptional activity according to this binding has apparently only evolved in chordates [3] The physiological role of AhR in vertebrates has not yet been completely elucidated, but it is known to regulate specific phase I and II metabolic enzymes, among others [4,5] Ectopic activation of AhR constitutes an initial step leading to toxic effects of a variety of harmful pollutants, such as 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) and benzo[a]pyrene [6,7], which include immune dysfunction, endocrine disruption, reproductive toxicity, developmental defects, and cancer in vertebrates [8–12] The use of yeast systems to monitor the interaction of different chemicals with vertebrate receptors has become a common tool to detect the presence of receptor-binding activity in the environment [13–16] These yeast-based bioassays, known as recombinant yeast assays (RYAs), have been used to correlate the presence of suspected or bona-fide endocrine disruptors and estrogenic activity in environmental samples [17– 19], and to establish relationships between chemical structures and affinity for vertebrate hormone receptors [20–24] It is generally accepted that ligand-free AhR molecules are mainly cytoplasmic, and that binding to the ligand triggers the translocation of the receptor–ligand complex to the cell nucleus During this process, the receptor–ligand complex binds to an auxiliary cofactor, the AhR nuclear translocator (ARNT), to form a ternary complex, which is capable of recognizing specific DNA sequences (xenobiotic responsive elements; XRE) in the promoter of target genes, increasing their transcription rates [25] Both AhR and ARNT by themselves are capable of triggering transcription when tethered to upstream regions of reporter genes by heterologous DNA binding domains (DBD) [26,27] More specifically, the ligand-binding domain (LBD) of AhR, when fused to an heterologous DBD, produces a ligand-dependent, ARNT-independent activator maintaining most pharmacological features of the AhR ⁄ ARNT ⁄ XRE system [27] These chimeric systems have been used mostly in yeast, but they also work in mammalian cell lines [28] 3328 The mechanisms by which transcriptional activation occurs upon binding of the ligand ⁄ AhR ⁄ ARNT complex to XRE are still unclear, but they probably include recruiting of different coactivators and general transcription factors, which ultimately promote transcription initiation by interacting with the RNA polymerase II [29] A key component of this mechanism is the cAMP response element-binding protein (CBP) ⁄ p300 complex, which is assumed to have a major role on the transcriptional activation by AhR ⁄ ARNT in mammals by interacting with histone acetyltransferases (HATs) [29,30] In yeast, a key coactivator for ligand-dependent transcriptional activation by AhR is the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex [31], a HAT complex required for function in yeast of many, but not all, transcriptional activators, including Gcn4p and VP16 [32,33] It is also required for liganddependent activation mediated in yeast by several vertebrate receptors, including the glucocorticoid, estrogen and retinoic acid receptors [34–36] Carbaryl (1-naphthyl-N-methylcarbamate) is a widespectrum carbamate insecticide that has been applied for approximately 40 years as a contact and ingestion insecticide on a wide variety of crops, as well as on poultry, livestock and pets It is also used as acaricide and as molluscicide in aquaculture facilities It has been reported that this compound is an inducer of cytochrome P450 1A gene expression [37,38], a biomarker of ectopic activation of the AhR receptor by exogenous ligands present in the environment [11,39] However, carbaryl differs structurally from typical AhR ligands, which are aromatic compounds with two or more rings in the same plane that can be accommodated within a rectangular binding site of approximately ˚ ˚ ˚ 14 A · 12 A · A [40] Carbaryl does not fit easily into these structural constraints (Fig 1) Nevertheless, both activation of the AhR system by carbaryl in cultured mammalian cells and specific binding in vitro of carbaryl to AhR has been demonstrated [41] The present study intended to further characterize the interaction of carbaryl with AhR by using a combination of Fig Chemical structures of b-naphthoflavone (left) and carbaryl (right) FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS S Boronat et al mammalian cell culture and yeast-based systems The data obtained suggest that the peculiar structure of carbaryl imposes a nonstandard structure of the AhRLBD, which in turns modulates the capacity of the complex to interact with CBP ⁄ p300 or SAGA This property of carbaryl may be relevant in the explanation of its physiological effects, and provide an explanation for the largely contradictory current data on the effects of carbaryl in different cell lines and tissues Results Differential response of pLMAX and YCM systems to carbaryl Addition of increasing concentrations of carbaryl to YCM cells resulted in a bell-shaped activation ⁄ toxicity curve, as described for many receptor agonists that become toxic or inhibitory at high concentrations [22] (Fig 2) The calculated EC50 value for carbaryl in YCM cells was 124.3 ± 9.6 lm (Table 1), which corresponds to a weak agonist At higher concentrations, carbaryl becomes an inhibitor, with an apparent IC50 value of 578.0 ± 36.2 lm (Table 1) By contrast, carbaryl was unable to activate the LMAX-RYA system at any concentration (not shown) In this system, carbaryl acted as an antagonist because simultaneous addition of lm of a typical AhR ligand, b-naphthoflavone (BNF), and increasing concentrations of carbaryl resulted in a typical inhibition curve, with an apparent IC50 of 256.3 ± 38.2 lm (Fig and Table 1; see BNF and carbaryl structures in Fig 1) Therefore, the response Modulation of AhR transactivation by carbaryl Table Adjustments to the different activation ⁄ inhibition models a 95% confidence margins Model System Equation EC50 ⁄ IC50 (lM)a Activation Inhibition Inhibition Competitive inhibition Irreversible inhibition Toxicity YCM-RYA YCM-RYA LMAX-RYA LMAX-RYA LMAX-RYA Gal-GUS Eqn Eqn Eqn Eqn Eqn Eqn 124.3 578.0 256.3 77.3 461.5 459.8 (1) (1) (1) (2) (3) (3) ± ± ± ± ± ± 9.6 36.2 38.2 15.3 102.3 74.3 to carbaryl depended on the RYA system used and, presumably, on the transcriptional activation mechanism predominant in each of them Carbaryl as a competitive inhibitor of AhR in yeast To elucidate the mechanisms causing inhibition of carbaryl in LMAX-RYA, a number of dose–response assays with increasing concentrations of BNF were performed in the presence of different concentrations of carbaryl As shown in Fig 3A,B, the presence of carbaryl affected both the maximal activation at saturating concentrations of BNF and the position of the sigmoidal curve Whereas the latter is consistent with a competition for binding to AhR-LBD by carbaryl and BNF, the decrease of the maximal activation value is more consistent with a noncompetitive inhibition, either reversible or irreversible IC50 values were obtained for both effects separately (Table 1) from the analysis of the experimental data shown in Fig 3A,B Figure 4A shows the adjustment of the apparent EC50 values at different carbaryl concentrations to a competitive inhibition model [Eqn (2)] An IC50 value of 77.3 ± 15.3 lm can be calculated from the slope of the regression line (Table 1) Similarly, the decrease of maximal activation at increasing concentrations of carbaryl can be adjusted to a noncompetitive binding model [Eqn (3); Fig 4B] In this case, the corresponding IC50 value obtained from the slope of the regression line was significantly higher, 461.5 ± 102.3 lm (Table 1), which is compatible to the toxic effect observed in YCM-RYA Therefore, we conclude that the behaviour of carbaryl in both RYA systems is similar, with a binding constant of approximately 100 lm and a toxic effect at concentrations higher than 400 lm Analysis of carbaryl toxicity in yeast Fig Dose–response curve for carbaryl in YCM-RYA (s) and in LMAX-RYA with simultaneous addition of lM BNF (d) Data are the average of four independent determinations; bars represent standard errors Irreversible inhibition can also be explained as a consequence of cell inactivation by the inhibitory ligand In this case, the phenomenon would not be related to the FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3329 Modulation of AhR transactivation by carbaryl S Boronat et al A B Fig Data adjustment for the competitive-reversible (A) and irreversible (B) models for the experiments in Fig 2; IC50 values were calculated from the slopes of the regression lines (- - -) Fig Dose–response of BNF in the presence of increasing concentrations of carbaryl in pLMAX-RYA (A) Showing data relative to the maximal activity of BNF in the absence of carbaryl Data were adjusted to the maximal concentration in each series (relative expression) (B) Showing unadjusted data (GUS arbitrary units) Dots represent replicas for each series; curves are calculated from the observed EC50 for each carbaryl concentration (all replicas combined) characteristics of the receptor, but to the sensitivity of the particular cell strain used in the assay This effect can be monitored by measuring the effect of the compound to the activation of galactose-responsive genes, an endogenous yeast activation mechanism completely unrelated to AhR [42] Figure shows the decrease of 3330 Fig Inhibition of galactose response by carbaryl in GAL-GUS system The discontinuous curve represents a nonlinear fitting to a logistic function cell response to galactose in the presence of increasing concentrations of carbaryl The decrease follows a sigmoidal curve with an IC50 value of 459 ± 74 lm for carbaryl, similar to the Ki value obtained for the FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS S Boronat et al Modulation of AhR transactivation by carbaryl negative effect at high concentrations of carbaryl in both RYA systems (irreversible model; Table 1) Therefore, we consider that the noncompetitive ⁄ irreversible component of carbaryl inhibition in LMAXRYA was likely due to cytotoxicity rather than to a putative second site for carbaryl binding in the AhR Carbaryl as a competitive inhibitor of AhR in vertebrate cell cultures Two vertebrate cell lines were tested for their sensitivity to the presence of carbaryl in dose–response assays using BNF as agonist The CALUXÒ cell line, commonly used for testing AhR agonists, showed essentially identical dose–response curves in the presence and absence of 200 lm carbaryl, with EC50 values of 8.40 ± 1.32 lm and 9.04 ± 1.34 lm for BNF, respectively (Fig 6) By contrast, the rainbow trout liver (RTL) cell line showed an EC50 for BNF of 0.75 ± 0.28 lm, approximately one tenth of the corresponding value for CALUXÒ These values increased to 4.25 ± 0.87 lm when the dose–response curve was performed in the presence of 200 lm carbaryl, indicating an antagonistic effect in these cells similar to the one observed for the yeast YCM-RYA system (Fig 6; compare with Fig 3) Modulation of the interaction of AhR-LBD with CBP by AhR ligands AhR activation is at least partially mediated by the recruitment of CBP to the target promoters [29] To assess the influence of different ligands in this interaction, we performed two-hybrid assays in yeast, using the pLMAX as a DNA binding domain and CBPGal4AD as activation domain Addition of different concentrations of BNF to the triple-transformant resulted in a significant, 50% increase of maximal transcription level relative to an isogenic strain lacking the CBP-Gal4AD plasmid (Fig 7A) This effect was minimal, if any, when carbaryl was added to the same strain, indicating that the interaction between carbarylloaded AhR-LBD and CBP did not occur (Fig 7B) The effect of the presence of CBP-Gal4AD in the two-hybrid system was obscured by the strong activation signal of pLMAX-RYA in the presence of BNF This activity can be strongly reduced by the disruption of the endogenous gene ADA2 in yeast [32] (Fig 7C, compare fluorescence units with Fig 7A) Dada2 strains expressing pLMAX showed a limited response to the presence of BNF, and no response whatsoever to carbaryl (Fig 7C,D) In this specific genetic background, the presence of the CBP-Gal4AD construct increased Fig Dose–response curves for BNF in the presence (s) and absence (r) of 200 lM carbaryl in (A) RTL cells and (B) DR-CALUXÒ system Values are average of three independent determinations; bars represent standard deviations transcriptional response to BNF by four to five-fold, whereas no significant response was observed when carbaryl was added (Fig 7C,D) As the activation potential of Gal4p activation domain present in the CBP-Gal4AD chimera is completely unrelated to the presence of AhR ligands, we conclude that the lack of response to carbaryl in the two-hybrid system was due to the inability of the carbaryl–AhR complex to interact with CBP At this point, it should be remembered that deletion of ADA2 affects very little the transcriptional activation by Gal4AD [43] Co-expression of CBPGal4AD In Dada2 strains did not increase expression of LexA-AhR-LBD The amount of LexA-AhR-LBD mRNA in Dada2 cells was calculated at 1.9 · 108 ± 8.5 · 107 copies per cell (an average of 12 independent FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3331 Modulation of AhR transactivation by carbaryl S Boronat et al Fig Dose–response curves for (A,C) BNF and (B,D) carbaryl in yeast strains transformed with pLMAX (—) or with pLMAX and pGADT7mCBP plasmids (- - - ), using pRB1155 plasmid as a reporter (A,B) Wild-type yeast strains; (C,D), Dada2 strains measurements) The corresponding figure for Dada2 cotransformed with pLMAX and pGADT7-mCBP was 1.3 · 108 ± 7.1 · 107 mRNA copies per cell (12 determinations) These two values were not statistically different (P > 0.05, Student’s t-test); therefore, we attributed the increased response to BNF in CBPGal4AD expressing Dada2 strains to a higher efficiency to promote transcription of the LexA-AhR-LBD ⁄ CBPGal4AD complex relative to the LexA-AhR-LBD alone, rather than to a differential expression of the LexA-AhR-LBD fusion protein From these data, we conclude that there is ligand-dependent interaction between AhR and CBP upon addition of BNF, and that this interaction did not occur when carbaryl, instead of BNF, was added to the medium Discussion The molecular mechanisms underlying the activation of genes under the control of XREs by carbaryl have been object of controversy, especially because of contradictory reports on its ability to bind AhR [37,44–46] However, some recent determinations using computer modeling, together with experimental data from cell culture assays with DR-CALUXÒ (dioxin 3332 responsive-chemically activated luciferase) cells and from an immunoassay detecting activated AhR complexes, demonstrated that carbaryl can interact with the AhR and trigger transcriptional activation [41] The data presented here intend to further elucidate this mechanism by using a combination of mammalian cell culture and yeast-based systems, allowing the dissection of transcriptional activation pathways by genetic tools Carbaryl appears to be a better activator in YCMRYA [lowest observed effect concentration (LOEC), approximately 20 lm) than in the vertebrate DR-CALUXÒ system, with LOEC values of 100 lm [41] By contrast, carbaryl acted as a competitive antagonist, instead of as an agonist, in LMAX-RYA A similar antagonistic effect of carbaryl was observed in the mammalian RTL cell line, but not in the DR-CALUXÒ system, in which it is know to act as an agonist [41] We propose the peculiar structure of carbaryl as the main reason of this dual role as agonist and antagonist in yeast and cell culture mammalian systems In silico studies showed that carbaryl adopts preferentially nonplanar conformations, which, in principle, are less likely to interact with AhR, whereas even the most stable planar conformations are energetically slightly less favourable (less than kJỈmol)1) than FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS S Boronat et al Modulation of AhR transactivation by carbaryl noncoplanar ones [41] Assuming that interaction of carbaryl (as other AhR ligands) with AhR-LBD should occur through planar and close-to planarity conformers [41,47], the structural constraints of the resulting complexes could modify key surfaces of interaction with coactivators and preclude transcriptional activation This specific configuration of the AhRLBD would allow the translocation of the receptorligand complex to the nucleus and its interaction with ARNT, but not the interaction of AhR-LBD with transcriptional coactivators, including CBP, which are required for transcriptional activation In YCM-RYA, ARNT would provide for the missing interactions and therefore the system behaves as an agonist; in LMAXRYA these additional interactions would be missing and the resulting effect is competitive inhibition A simplified scheme of this model is depicted in Fig There are several reports in the literature of antagonists of AhR, including flavonoids [16,48,49] and several phenolic compounds, like resveratrol [50], either by inhibiting translocation of AhR to the nucleus and to stabilize the inactive AhR ⁄ hsp90 complex [48,49] or by inducing a inactive configuration to the ligand ⁄ AhR ⁄ ARNT ⁄ XRE complex [50] This latter mechanism may partially apply to carbaryl, with the difference that its binding to AhR may result in agonistic or antagonistic effects depending on the cofactors prevalent in each cell type, as illustrated by the different effects on RTL and DR-CALUXÒ cell lines The model proposed here is similar to the one proposed for some partial agonists of the estrogen receptor, such as tamoxifen [51] but, to our knowledge, ours is the first report indicating that it may also apply to AhR antagonists It is also the first one on proposing a specific AhR ⁄ coactivator interaction (CBP) as a target for AhR inactivation by a ligand The results presented here, together with other available data concerning gene activation by carbaryl in cell lines and in test animals, are relevant in predicting the effects of carbaryl when it is released into the environment Carbaryl exposure will likely result in the ectopic activation of the P450 system in vertebrates, although with less potency than other known pollutants However, this effect may vary in different tissues, and perhaps in different organisms, as the activation potential of activators may depend on the relative importance of key coactivators in different cell systems As the ectopic activation of P450 systems is considered to be detrimental in many biological systems [11], this argues for a stringent control of the release of carbaryl into the environment Experimental procedures Chemicals Carbaryl (Riedel-de Haen, Seelze, Germany) was obtained ă at a purity of 99.7% and BNF (used as positive control and considered to be a model ligand compound of the AhR) was obtained from Sigma (St Louis, MO, USA) at a minimum purity of 95% Stock solutions of both compounds were prepared by dissolving them in dimethyl sulfoxide (Sigma) Plasmids PLMAX Plasmid pLMAX contains a fusion construct between the LexA protein DNA binding domain (amino acids 1–202) and the 1914 bp EcoRI-XhoI fragment of the mouse AhR (amino acids 167–805) in the expression plasmid pLexA202 from Clontech (BD Biosciences, Palo Alto, CA, USA) YCM LMAX TCDD/βNF AhR Fig Model of transcriptional activation for TCDD ⁄ BNF (upper) and carbaryl (lower) in YCM-RYA (left) and LMAX-RYA (right) Note the difference on DNA binding domains and DNA sequences between both systems as well as the absence of ARNT in LMAX-RYA The proposed differential conformation of TCDD ⁄ BNF and carbaryl complexes with the AhR-LBD is also shown ARNT LexA-AhR LBD ARNT LexA-AhR LBD Carbaryl AhR FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3333 Modulation of AhR transactivation by carbaryl S Boronat et al Plasmid pGADT7-mCBP Recombinant yeast assay Plasmid pGADT7-mCBP was kindly provided by H Jiang [52] It contains the N-terminus of mouse CBP (amino acids 1–464) fused at the C-terminus of the GAL4 protein activation domain in the yeast expression vector pGADT7 from Clontech Yeast strains were grown overnight in minimal medium (6.7 gỈL)1 yeast nitrogen base without amino acids plus ammonium sulfate; DIFCO, Basel, Switzerland) supplemented with 0.1 gỈL)1 of prototrophic markers as required and with either glucose or galactose as a carbon source When cells were at the appropriate attenuance (0.1–0.2) they were mixed with carbaryl or with BNF dissolved in dimethyl sulfoxide Some 50–100 lL of this mix were added in triplicates in a 96-well siliconized polypropylene microtiter plate (NUNCTM, Roskilde, Denmark) and further diluted in the same plate in wells containing cell culture without the chemical Cells were incubated for h at 30 °C under mild shaking Permeabilization of yeast cells and fluorogenic quantitation of either lacZ or GUS activity was performed as described [16] EC50 values were calculated by fitting the data to a noncooperative version of the Hill equation using SPSS for Windows package (version 11.01, SPSS Inc Chicago, IL, USA), as described in [16] For general toxicity testing, the GAL-GUS system strain was grown overnight in minimal medium supplemented with 0.1 gỈL)1 of prototrophic markers as required and with raffinose as a carbon source When cells were at the appropriate attenuance (0.1–0.2), 2% galactose was added and they were mixed with carbaryl or BNF and treated as described above Plasmid pRB1155 Plasmid pRB1155 is a high copy number yeast reporter plasmid encompassing lexA-binding sites driving the expression of the lacZ reporter gene [53] Yeast strains and RYA systems AhR ⁄ ARNT system (YCM-RYA) Strain YCM4 was a generous gift from C A Miller (Tulane University, New Orleans, LA, USA) [54] This strain is a derivative of W303a (MATa, ade2-1, can1-100, his3-11, 15, leu2-3, 112, trp1-1, ura3-1), which harbours two foreign genetic elements: one of them is chromosomally integrated and coexpresses human aryl hydrocarbon receptor and ARNT genes under the GAL1-10 promoter The second construct is the pDRE23-Z reporter, encompassing three XRE5 sequence and the CYC1-lacZ fusion (more information in the original paper [54]) To perform the RYA assay, YCM4 and YCM4 derived cells were grown in galactose overnight to express both AhR and ARNT LMAX-system (pLMAX-RYA) YSB7 (MATa, leu2, his3, met 15, URA3::lexA-GUS) is a derivative of strain BY4741 (Euroscarf, Frankfurt, Germany) and contains the 2l plasmid pLMAX and eight copies of the LexA DNA recognition sequence in front of the b-glucuronidase (GUS) reporter gene integrated into the genome Strains YSB37 and YSB39 were constructed by transforming BY4741 and Y04282 (MATa, leu2, his3, met 15, ura3, ada2::kanMX4), obtained from Euroscarf, with plasmids pLMAX and pRB1155 Yeast strains YSB52 and YSB53 were obtained by transformation of YSB37 and YSB39 with plasmid pGADT7 RNA extraction and real time RT-PCR Total RNA was extracted using the MasterPureTM-Yeast RNA Purification kit from Epicentre Biotechnologies (Madison, WI, USA) and used according to the manufacturer’s instructions cDNAs were prepared with OmniscriptTM Reverse Transcriptase (Qiagen, Valencia, CA, USA) using oligo-dT primers and lg of total RNA as template lL of each cDNA and further : 10 dilutions were used for real time RT-PCR using SYBRÒGREEN PCR Master Mix (Applied Biosystems, Warrington, UK) with 300 nm of each primer in a final volume of 20 lL PCR was monitored in an ABIPrismTM 7000 Sequence Detection System (Applied Biosystems), using the following primers: 5¢-AGTTTTCCGGCTTCTTGCAA-3¢ (forward) and 5¢-TTGGACTGGACCCACCTCC-3¢ (reverse), from Roche (Basel, Switzerland) LexA-mAhR-LBD mRNA copy numbers were calculated by interpolation in a standard curve using plasmid pLMAX as standard GAL-GUS system A yeast reporter strain was constructed, in which GUS transcription was controlled by the GAL1-10 promoter [42] Briefly, yeast strain BY474 was transformed by one-step double homologous recombination using two overlapping PCR fragments that allowed both GUS integration at the GAL1,10 site and nourseothricin selection Details about the strategy and the characterization of the strain are provided elsewhere [55] 3334 Vertebrate cell lines culture and enzymatic measurements The rainbow trout liver cell line, RTL-W1, was grown as outlined in the original description of this cell line [56] Cells were grown in Leibovitz’s L-15 cell culture medium (Cambrex, North Brunswick, NJ, USA) supplemented with FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS S Boronat et al 5% fetal bovine serum (Cambrex) and penicillin–streptomycin (20 mL)1 to 20 lgỈmL)1, respectively, Cambrex) in 75 cm2 NUNCTM tissue-culture flasks (Nalgene Nunc International, Rochester, NY, USA) at 19 °C Cells were detached from confluent flasks using trypsin (Sigma), and then, seeded in 96 well Falcon plates (Becton Dickinson, Oxnard, CA, USA) at a density of 20 000 cells in 200 lL of culture medium per well and allowed to grow to confluency for day Subsequently, medium was substituted and new medium with the corresponding concentrations of BNF (0.2–100 lm) and carbaryl (100 lm) was added The maximal concentration of dimethyl sulfoxide in the culture medium was 0.2% Control cells received only solvent After 48 h of treatment, medium was removed, cells washed with phosphate buffered saline (pH 7.5) and the plates frozen in liquid nitrogen They were maintained at )80 °C until analysis of ethoxyresorufin-O-deethylase activity and protein following the methodology previously described [57,58] The DR-CALUXÒ bioassay performed in this study is based on the use of a rat hepatoma (H4IIE) cell line stably transfected with a construct containing the luciferase reporter gene under direct control of DRE (Dioxin Responsive Element) (BioDetection Systems, Amsterdam, the Netherlands) Cells were maintained in aMEM (Cambrex) with phenol red and supplemented with 10% fetal bovine serum (Cambrex), 1% mm l-glutamine (Cambrex) and penicillin–streptomycin (10 mL)1 to 10 lgỈmL)1, respectively, Cambrex) Cells were grown at 37 °C with 5% CO2 in a humidified incubator For the assay, cells grown in bottles were trypsinized and plated in 96 well plates at a density of 2.5 · 104 cells per well After 24 h, the cells were cotreated with different concentrations of BNF (0.3–100 lm) and a fixed concentration of carbaryl (200 lm) Carbaryl or BNF stock solutions were diluted in culture medium at a maximal solvent concentration of 0.2% Control cells received the maximal dimethyl sulfoxide concentration used in the treated cells Cells were exposed to the xenobiotics for 48 h Subsequently, culture plates were washed with phosphate buffer and the luminescence emitted by the cells was quantified by means of the SteadyGlo Luciferase assay System from Promega (Madison, WI, USA) following the manufacturer’s instructions in a Tecan Genios (Maennedorf, Switzerland) luminescence detector Mathematical modelling The equations and definitions used in this work are derived from standard ligand-receptor mathematical models, as previously described [59] A more detailed description of the models can be found in the Supplementary material Modulation of AhR transactivation by carbaryl Interaction of a receptor with a single ligand The simplest model to describe dose ⁄ response curves assumes an equilibrium between hormone-free and hormone-loaded hormone receptor molecules in solution: Kd R ỵ h1 Rh1 where R represents the concentration of hormone-free receptor molecules, h is the hormone concentration, Rh is the concentration of the hormone-loaded receptor molecule, and Kd is the dissociation constant The model assumes a single agonist molecule binding to each receptor molecule, and an hormone concentration much larger than the receptor concentration The fraction of receptor bound to the hormone Fr can be described by the Hill equation: Ur ẳ ẵRh ẳ Ro ỵ Kd ẵh ẵ1 In which Ro is the total receptor concentration (bound and free) From Eqn (1), Kd can be calculated as the ligand concentration at which 50% of receptor molecules are occupied, which in turn coincides with EC50, the hormone concentration at which the physiological effect (i.e the reporter activity in our case) reaches 50% of its maximal value at saturating hormone concentration When applied to inhibitory effects, such as a decrease on transcription rates upon addition of a compound, the equilibrium constant is usually denominated as Ki and its value coincides with IC50, the effector concentration at which the measured physiological activity is reduced to 50% Interaction of a receptor with two ligands Below, we considered three mechanisms of mutually interaction among a pure agonistic ligand (h1, in our case, BNF) and an inhibitor (h2, in our case carbaryl) Reversible binding, competitive inhibition This model proposes an equilibrium between free receptor, R, and two ligands that bind alternatively to a single site of the receptor molecule, with dissociation constants Kd1 and Kd2 : Kd1 Kd2 R ỵ h1 Rh1 ; R ỵ h2 Rh2 At any given concentrations of h1 and h2, any target gene would show a fraction of its maximal activation at saturating concentration of h1 A ⁄ Amax that could be expressed as: A ẵRh1 ẳ ẳ Amax Ro Kd ẵh 1ỵ Kd1 ỵ Kd 2 ½h1 Š In this variant of the Hill equation, Amax is independent from h2, whereas the apparent EC50 for h1 (EC50app) equals FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3335 Modulation of AhR transactivation by carbaryl S Boronat et al to Kd1 only when h2 ¼ Kd2 (identical to IC50) can be calculated by measuring EC50app at different concentrations of h2 following the equation: EC50h2 ị ẵh2 ẳ1ỵ Kd2 EC50h2 ẳ0ị Rh2 ẳ K ; Ro ỵ ẵh2i R ¼ Ro À Rh2 In this model, the maximal activity at saturating concentrations of h1 depends on the concentration of h2, as follows: ½2Š h1 ! 1; in which EC50h2 ị and EC50h2 ẳ0ị correspond to the EC50 for h1 in the presence and in the absence of a given concentration of h2 Noncompetitive, reversible inhibition This model postulates the binding of h1 and h2 to two independent binding sites in the receptor, and that binding of h2 allows binding of h1 but precludes transcriptional activation The model predicts three ligand-receptor complexes: Amax; h2 ẳ0 Ro ẵh2 ẳ1ỵ ¼ Ki Amax; h2 R Ki is therefore equivalent to IC50 This equation is identical to Eqn (3), and therefore Ki can be calculated as Kd2 in the previous model Acknowledgements This work has been supported by the Spanish Ministry for Science and Technology (BIO2005-00840) and INIA (RTA2006-00022-00-00) The contribution of the ` Centre de Referencia en Biotecnologia de la Generalitat de Catalunya is also acknowledged References The fraction of the active complex relative to the total amount of receptor molecules, Ro can be calculated as: ẵRh1 ẳ Ro ẵh2 ỵ Kd2 ỵ ẵh Kd1 1ỵKd ị ẵh1 The model predicts that the apparent EC50 for h1 is independent from the concentration of h2, and therefore identical to the calculated Kd1 in the absence of h2 However, at saturating concentrations of h1, the maximal response given by the system, Amax, depends solely on 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from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article The following supplementary material is available online: Doc S1 The equations and definitions used in this work are derived from standard ligand-receptor FEBS Journal 274 (2007) 3327–3339 ª 2007 The Authors Journal compilation ª 2007 FEBS 3339 ... Alonso M, Herradon B, Tarazona J & Navas J (2006) Activation of the aryl hydrocarbon receptor by carbaryl: computacional evidence of the ability of carbaryl to assume a planar coformation Env Toxicol... Nishihara T, Nishikawa J, Kanayama T, Dakeyama F, Saito K, Imagawa M, Takatori S, Kitagawa Y, Hori S & Utsumi H (2000) Estrogenic activities of 517 chemicals by yeast two-hybrid assay J Health... transcriptional activation by Gal4AD [43] Co-expression of CBPGal4AD In Dada2 strains did not increase expression of LexA-AhR-LBD The amount of LexA-AhR-LBD mRNA in Dada2 cells was calculated at