Aryl Hydrocarbon Receptor Activity of Polyhalogenated Carbazoles and the Molecular Mechanism

Science of the Total Environment 687 (2019) 516–526

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Science of the Total Environment

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Aryl hydrocarbon receptor activity of polyhalogenated carbazoles and the molecular mechanism

Dan Ma a,b, Heidi Qunhui Xie a,b, Wanglong Zhang a,b,QiaoXuea,b, Xiuchang Liu a,b,LiXua,b, Yongchao Ma a,b, Eva Cecilie Bonefeld-Jørgensen c, Manhai Long c,AiqianZhanga,b,BinZhaoa,b,⁎ a State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center of Eco-Environment Sciences, Chinese Academy of Sciences, Beijing 100085, China b University of Chinese Academy of Sciences, Beijing 100049, China c Centre for Arctic Health & Molecular Epidemiology, Department of Public Health, Aarhus University, Aarhus DK-8000, Denmark

HIGHLIGHTS GRAPHICAL ABSTRACT

• Nine PHCZs concentration-dependently activated AhR via luciferase reporter assay. • The 9 PHCZs signiﬁcantly induced CYP1A1 gene expression. • The 9 PHCZs could be docked into the same pocket as TCDD. • The docking poses of 2367-CCZ and 27- BCZ were distinct from the others. • Two types of antagonizing effects of CH223191 were found accordingly.

article info abstract

Article history: Polyhalogenated carbazoles (PHCZs) are a class of contaminants identiﬁed with persistence and bioaccumulation Received 10 April 2019 property from previous studies. However, the toxic effect and mechanism of PHCZs are not fully understood. In Received in revised form 24 May 2019 this study, eleven PHCZs, including four chlorocarbazoles, four bromocarbazoles and two bromo/ Accepted 26 May 2019 chlorocarbazoles were screened for their potential aryl hydrocarbon receptor (AhR) activity by using a dioxin re- Available online 30 May 2019 sponsive element-driven luciferase reporter assay. We found that nine PHCZs signiﬁcantly activated AhR in a Editor: Jay Gan concentration-dependent manner. Their potencies of AhR activation were 1000 to 100,000 folds less than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand. The relative AhR activation potency Keywords: of the nine PHCZs followed the order 2,3,6,7-tetrachloro-9H-carbazole N2,7-dibromo-9H-carbazole N1,3,6- Polyhalogenated carbazoles tribromo-9H-carbazole N1,3,6,8-tetrachloro-9H-carbazole N1,3,6,8-tetrabromo-9H-carbazole N1-bromo-3,6- Aryl hydrocarbon receptor dichloro-9H-carbazole N3,6-dibromo-9H-carbazole N3-bromo-9H-carbazole N1,8-dibromo-3,6-dichloro-9H-car- CH223191 bazole, which was partly in line with the induction of AhR-mediated CYP1A1 expression. In silico analysis indi- 2,3,7,8-tetrachlorodibenzo-p-dioxin cated that the nine PHCZs could be docked into the same pocket as TCDD due to their high structural Luciferase reporter assay similarity. However, the shrunk size of the heterocyclic moieties in PHCZs relative to that in TCDD dramatically decreased the complex stability provided by inter-molecular interactions. Moreover, two distinguished docking poses adopted by the nine PHCZs were found, in which one was illustrated by 2367-CCZ and 27-BCZ while the other symbolized by TCDD and the left seven agonists. The differential antagonizing effects of CH223191 on PHCZ-induced AhR activity supported such pose differentiation. The present experimental and in silico data

⁎ Corresponding author at: Research Center for Eco-Environmental Sciences, (RCEES), Chinese Academy of Sciences, Beijing 100085, China. E-mail address: [email protected] (B. Zhao).

1. Introduction human breast cancer cells and rat hepatoma HII4E cells (Lisa et al., 2011; Mumbo et al., 2015; Riddell et al., 2015). Thus, AhR-dependent Polyhalogenated carbazoles (PHCZs) were firstly reported by Kuehl pathway has been proposed as one of the possible molecular mecha- et al. in the 1980s who advertently identified this group of compounds nisms for PHCZ-induced CYP1A1 induction. However, there is still a while determining other persistent organic pollutants (POPs) in the lack of direct evidence demonstrating the interaction between PHCZs sediment from Buffalo River (Kuehl et al., 1984). Since then, PHCZs and AhR. have been detected in various environmental matrices widely distribut- As a member of basic helix-loop-helix/Per-Arnt-Sim (PAS) super- ing in the world, including lake sediments (Guo et al., 2016; Wu et al., family (Furness and Whelan, 2009), AhR is a ligand-activated transcrip- 2016), marine shore sediments (Grigoriadou and Schwarzbauer, tion factor and involved in many important physiological processes 2011a), soils (Mumbo et al., 2016), water (Grigoriadou and such as regulation of cell cycle proliferation, immune responses, circa- Schwarzbauer, 2011b), algae (Lee et al., 2010)andfish (Wu et al., dian rhythm (Pendergast and Yamazaki, 2012), tumor promotion (Ray 2018). In terms of different halogen substitutions, PHCZs consist of and Swanson, 2009), lipid metabolism-associated genes expression chlorocarbazoles, bromocabazoles, bromo/chlorocarbazolesas as well (Gouédard et al., 2004a) etc. Under quiescent condition, AhR exists in as less frequently determined iodocarbazoles. Both anthropogenic and cytosol, and the translocation into the nucleus is triggered upon its acti- natural sources contribute to the occurrence of PHCZs. Certain PHCZs vation by ligand-binding and the subsequent disassociation from chap- occurred more frequently in the Great Lakes as unintentional by- erone proteins (Zhou, 2016). The AhR nuclear translocator (ARNT) then products of indigo dyes industry (Parette et al., 2015). For instance, binds to the activated AhR to form a heterodimer which subsequently 1,3,6,8-tetrabromo-9H-carbazole (1368-BCZ) could be formed during binds to specific DNA sequences called dioxin response element (DRE) the production of 5,5′,7,7′-tetrabromoindigo while 1,8-dibromo-3,6- (Beischlag et al., 2008). A battery of genes including the CYP1A subfam- dichloro-9H-carbazole (18-B-36-CCZ) is originated from 7,7′-dibromo- ily are regulated by the activation of AhR-dependent pathway (Garrison 5,5′-dichloroindigo manufacture. Besides, 2,7-dibromo-9H-carbazole et al., 1996; Wu et al., 2018; Zhang et al., 2019). Various environmental (27-BCZ), 3,6-dibromo-9H-carbazole (36-BCZ) and 3,6-diiodo-carba- pollutants or natural chemicals with diverse structures have been iden- zole were found as natural products in marine heterocystous cyanobac- tified as AhR ligands including dioxins (Olivier, 2014), polychlorinated terium in Hong Kong shore area which may be related to the nitrogen- biphenyls (Nault et al., 2012), ginsenosides (Hu et al., 2013), flavones fixing ability of this species (Lee et al., 2010). Mumbo et al. reported that (Wang et al., 2018), etc. Among these compounds, 2,3,7,8- in the presence of chloroperoxidase, bromo- and chlorocarbazoles can tetrachlorodibenzo-p-dioxin (TCDD) is known as one of the most po- be enzymatic synthesized from Caldariomyces fumago in water tent AhR ligands. Several AhR-based bioassays have been used for ligand (Mumbo et al., 2013). identification and evaluating the relative potency of AhR activation (Shu Emerging evidence has suggested that certain PHCZs may possess et al., 2003; Vondráček et al., 2016; Weber and Greim, 1997). Among POPs properties. The log value of octanol-water partition coefficient (log them, DRE-driven luciferase reporter assay has been utilized frequently Kow) of some PHCZs have been predicted (Mumbo et al., 2015). Certain which is one of the most sensitive assays (Denison and Nagy, 2003; Lee di-, tri- and tetra-BCZs and -CCZs were high hydrophobic (log Kow ≥ 5), et al., 2015; Zhang et al., 2018). indicating that they could be bioaccumulated through the food chain. A In order to reveal the interaction between PHCZs and AhR and ex- persistence of N120 days was recorded for 3-chloro-9H-carbazole (3- plore the underlining molecular mechanism, we employed a newly CCZ) and 3,6-dichloro-9H-carbazole (36-CCZ) which are above the per- established and highly sensitive recombinant mouse hepatoma cell sys- sistent substance criterion set by European Commission chemical legisla- tem, which has been integrated with a novel DRE-driven luciferase re- tion on Registration, Evaluation, Authorization and Restriction of porter, namely CBG 2.8D. In CBG 2.8D cells, the luciferase activity was Chemicals (ECHA, 2017; Mumbo et al., 2015). Nitrogen-containing het- gradually increased upon 4-h to 24-h treatment with TCDD implying a erocycle as well as polychlorinated aromatic hydrocarbons of PHCZs stable responsiveness of reporter gene during the time (Zhang et al., have been proposed as key structure features for the resistance to natural 2018). By using the DRE-driven luciferase reporter system in CBG 2.8D degradation (Lundstedt et al., 2010; Wild et al., 1991). Besides, 1368-BCZ cells, we aim to (1) screen potential AhR ligands among eleven PHCZs was found to be resistant to degradation in sediment (Lingyan and Hites, which have been frequently identified in environmental samples, and 2005), and certain PHCZs was found to be bioaccumulated in fish from the revealing their relative AhR activation potency compared to TCDD; Great Lakes (Wu et al., 2018). However, the photo degradation of 3- (2) examine the effect of a ligand-selective AhR antagonist on PHCZ- bromocarbazole (3-BCZ), 3-CCZ, 36-BCZ and 36-CCZ has been identified, AhR interaction. To further verify the direct PHCZ-AhR interaction iden- and direct photolysis followed first-order kinetics has been recognized tified through the reporter system, in Silico analysis was carried out, and in ultrapure water system. Since the maximum wavelengths of absorp- potential residues responsible to interaction and mode of action were tion cover near ultraviolet region (295, 296, 299, and 301 nm), these com- proposed. Meanwhile, mRNA expression of CYP1A1, a typical respon- pounds have been proposed to degrade slowly under sunlight in natural sive gene of AhR signaling pathway, was determined after treatment aquatic environment. (Mumbo et al., 2017). with AhR activating PHCZs in order to further support the action on Due to their persistence and bioaccumulative properties, PHCZs are AhR signaling pathway. worthy of close concern in ecology and toxicology. However, the knowl- edge about their potential harmful effects on animals or human-beings 2. Materials and methods is still limited. Given similar structure to polyhalogenated dibenzo-p-dioxin, the potential toxicity of PHCZs was examined during zebrafish de- 2.1. Cell culture velopment, and similar cardiotoxicities to dioxin were identified (Fang et al., 2016). Moreover, several pieces of evidence have shown that CBG 2.8D, a stably transfected mouse hepatoma cell line carrying the the expression of one downstream responsive gene of aryl hydrocarbon DRE-driven luciferase reporter gene, was established by our own labo- receptor (AhR) pathway, cytochrome P450 (CYP) 1A1 and its enzymatic ratory previously (Zhang et al., 2018). Cells were seeded in 100 mm activity could be induced upon PHCZs exposures in MDA-MB-468 cell-culture dishes and grown in α-modified Eagle's minimum essential 518 D. Ma et al. / Science of the Total Environment 687 (2019) 516–526 medium (α-MEM, Invitrogen, Carlsbad, CA) supplemented with 10% 2.5. Homology modeling and docking analysis fetal bovine serum (FBS, Invitrogen) and 1% penicillin–streptomycin

(Invitrogen). The incubator was setting at 37 °C with 5% CO2 and satu- The encoding sequence for mouse AhR (mAhR) ligand binding do- rated humidity. main (LBD) (Sequence ID: NP_001300956.1) was obtained from the National Center for Biotechnology Information (NCBI). SWISS-MODEL 2.2. Chemical treatment was used to construct 3D structure of mAhR-LBD (Fig. S1) (Scheuermann et al., 2009; Waterhouse et al., 2018). The query cover- CBG 2.8D cells were plated into white clear-bottom 96-well cell- ages of the HIF2α PAS-B domain in the template, a crystal structure of culture plates (Corning, Oneonta, NY, USA) or 6-well cell-culture plates the high affinity heterodimer of HIF2α and ARNT C-terminal PAS do- (Corning) at 400,000 cells/mL for luciferase reporter gene assay or mains (PDB ID 3F1O), was 31% (Fig. S2) (Scheuermann et al., 2009). mRNA evaluation, respectively. After 24 h incubation, cells were treated Qualitative Model Energy ANalysis (QMEAN), a composite scoring with solvent control dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. function for validating and describing the quality of full protein struc- Louis, MO, USA), TCDD (Wellington Laboratories Inc., Ontario, Canada) tures as well as on a local per residue scale, was used to estimate the dissolved in DMSO at concentration of 1.5E-13 mol/L—1.0E-9 mol/L, or quality of the generated mAhR-LBD model through the SWISS-MODEL indicated PHCZs all dissolved in DMSO (Table 1) (Wellington Laborato- server (Fig. S3). Besides, Ramachandran plotwas also generated to ries Inc.) at various concentration series (2367-CCZ: 6.8E-12 mol/L— check the rationality of the secondary structures of the obtained LBD 6.8E-6 mol/L; 3-BCZ: 2.0E-12 mol/L—2.0E-6 mol/L; 1368-CCZ, 27-BCZ, structure through combination of phi (Φ)andpsi(Ψ)angles 36-BCZ, 1-B-36-CCZ: 1.5E-12 mol/L—1.5E-6 mol/L; 136-BCZ, 1368- (Fig. S4). Finally, the LBD pocket corresponding to the large internal BCZ, 18-B-36-CCZ: 1.0E-12 mol/L—1.0E-6 mol/L; with an order of mag- cavity that accommodates ligands in the HIF2α PAS-B domain was nitude as the interval) for luciferase reporter assay or 1E-6 mol/L for selectedfor docking analysis, and the cubic cavity of 40 Å × 40 Å CYP1A1 gene expression assay for another 24 h at 37 °C. No obvious dif- × 40 Å was adopted. The binding affinities for both TCDD and the ference in cell viability (90—110% of control level) was observed in tested PHCZs were obtained using AutoDock Vina (Oleg and Olson, PHCZs treated GBG 2.8D cells compared to solvent treated cells. While 2010). Further computation was carried out on the obtained docking determining the effect of AhR antagonist CH223191 (1-Methyl-N-[2- complex to ensure the binding pose of the ligands using energy mini- methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5- mization and molecular dynamics along with an AMBER7 FF99 force carboxamide; Sigma-Aldrich) on PHCZs-induced luciferase activity field as described in the previous work (Mu et al., 2011) (Zhao et al., 2010), cells were pre-incubated with CH223191 (1E- 7 mol/L, 1E-6 mol/L or 1E-5 mol/L in DMSO) 3 h before each PHCZ treatment. For single exposure experiment, the final DMSO concentration in 2.6. Evaluation of AhR activity and relative effect potency (REP) all treatment and control groups were 1%; for CH223191 co-exposure experiment, the final DMSO concentration in all treatment and control The half maximum effective concentrations (EC50)werepre- groups were 1.1%. dicted by fitting dose-response data to a sigmoidal equation with 95% confidence intervals for AhR activity evaluation (Li et al., 2.3. Luciferase reporter assay 2005). Relative effect potency of AhR activation was estimated based on a systematic framework proposed by Villeneuve et al. The luciferase activity measurement was performed using a lucifer- (Villeneuve et al., 2010). First, the maximal fold of induction of ase reporter assay system kit (Promega, Madison, WI, USA). Briefly, the TCDD compared by DMSO solvent control was obtained, namely in- cells were rinsed twice with 100 μLofphosphate-bufferedsaline(PBS, duction fold of positive control (PC). Then 10%, 20%, 50%, or 80% of pH 7.4) after medium removal. Then 100 μL of cell-lysis buffer the maximal fold of TCDD induction (PC value) were calculated,

(Promega) was added into each well following a 15 min shaking at namely PC10,PC20,PC50,orPC80, respectively. Then the concentra- room temperature. Measurement of luciferase activity was carried out tions of TCDD and each test PHCZ that elicited the same induction using a microplate luminometer (GlomaxMulti Plus, Promega) with au- fold as that of corresponding PCx were ﬁgured out using interpola- tomatic injection of stabilized luciferase reagent (Promega). The optical tion method. REP was calculated as the following equation and the signal was collected as relative light unit (RLU) values. All samples were range of relative effect potency (REP) obtained based on the separate tested in triplicates. PCX values were displayed:

2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis ðÞ ¼ TCDD PCx REP ðÞ After total RNA extraction with GeneJET RNA purification kit PHCZ PCx (Thermo, Waltham, MA, USA), 2 μg RNA were used for cDNA synthesis with RevertAid first strand cDNA synthesis kit (Thermo). The qRT-PCR analysis was carried out using GoTaq® qPCR master mix kit (Promega) Where TCDD(PCx)andPHCZ(PCx) are the concentrations of TCDD with equal amounts of cDNA. All experiments were performed accord- and PHCZ respectively that elicited a response equal to 10%, 20%, 50%, ing to the manufacturer's instructions. The SYBR green signal was de- or 80% of the maximal fold of induction of TCDD. No range of REP values tected by a QuantStudion TM 6 Flex Real-Time PCR System (Thermo). was presented when only the REP of PC10 could be calculated. The results were analyzed by the ΔΔCT method (Hindiyeh et al., 2014). The housekeeping gene β-actin was used to normalize the target gene CYP1A1. For β-actin, the CT values in all groups were within a 2.7. Statistical analysis range of 18.76 ± 0.31 (mean ± SD, n =90),andthecoefficient of var- iation was 5.44%. No significant change in the β-actin expression was All the RLU values of treatment groups were divided by that of con- observed from all treated groups. Sequence of the primers used in trol (DMSO) groups for normalization. The data were presented as the qRT-PCR analysis was as follows: CYP1A1 (Sequence ID in GenBank: means ± SEM from three independent experiments. Statistical analysis NM_001136059.2) sense: 5′-CTCGTGGAGCCTCATGTACC-3′,antisense: and figure plotting were performed using GraphPad Prism software 5 5′-AACCTGCCACTGGTTCACAA-3′; β-actin (Sequence ID in GenBank: (GraphPad Software, USA). The differences between two groups were NM_007393.5) sense: 5′-TCATCACCATCGGCAACG-3′,antisense:5′- analyzed using an unpaired Student's t-test, and p b 0.05 (*) was consid- TTCCTGATGTCCACGC-3′. ered to be statistically significant. D. Ma et al. / Science of the Total Environment 687 (2019) 516–526 519

Table 1 Structure of relevant PHCZs and TCDD.

Compound/abbreviation Molecular structure

2,3,7,8-Tetrachlorodibenzo-p-dioxin TCDD

1,3,6,8-Tetrachloro-9H-carbazole 1368-CCZ

1,3,6,8-Tetrabromo-9H-carbazole 1368-BCZ

1-Bromo-3,6-dichloro-9H-carbazole 1-B-36-CCZ

2,3,6,7-Tetrachloro-9H-carbazole 2367-CCZ

(continued on next page) 520 D. Ma et al. / Science of the Total Environment 687 (2019) 516–526

Table 1 (continued)

Compound/abbreviation Molecular structure

1,8-Dibromo-3,6-dichloro-9H-carbazole 18-B-36-CCZ

3-Chloro-9H-carbazole 3-CCZ

3,6-Dichloro-9H-carbazole 36-CCZ

1,3,6-Tribromo-9H-carbazole 136-BCZ

2,7-Dibromo-9H-carbazole 27-BCZ D. Ma et al. / Science of the Total Environment 687 (2019) 516–526 521

Table 1 (continued)

Compound/abbreviation Molecular structure

3,6-Dibromo-9H-carbazole 36-BCZ

3-Bromo-9H-carbazole 3-BCZ

3. Results and discussion Based on the REP value, the 9 PHCZs was generally a class of weak AhR agonist when compared to TCDD. Comparing to the REP values of dioxins 3.1. AhR activity of the PHCZs and their REPs obtained previously in the same cell system, the AhR activation potency of 2367-CCZ, the most potent PHCZ congener in this study, is similar to the We examined the ability of eleven PHCZs to activate/inhibit AhR dioxins with lower REP values, such as 3,3′,4,4′,5-pentaCB with REP through a DRE-driven luciferase reporter gene assay, which is an AhR- value of 0.017 (Zhang et al., 2018). REPs for PHCZs which ranged from based bioassay and a sensitive method to detect dioxin and dioxin like 1.4E-6 to 6.0E-4 were lower than REPs of many dioxin-like compounds compounds (Long et al., 2013; Ghisari et al., 2015; Zhang et al., 2018). (Van den Berg et al., 2013). Since the AhR activation of 2367-CCZ was rel- The concentration-dependent luciferase gene expression proﬁles were ative stable after 12-h to 48-h treatment (Fig. S6C), the relative weak AhR obtained upon 24-h treatment with the eleven PHCZs and presented a activation of this compound might rather rely on the structure activity re- divergence in AhR activation. Among them, nine PHCZs signiﬁcantly ac- lationship. The position of halogen substitution is an important determi- tivated AhR in a concentration-dependent manner (Fig. 1). The EC50, nant of AhR activation. 2367-CCZ contains four lateral chlorine maximal observed effect concentration (MOEC) and maximal fold in- substituents comparable to its counterpart 2378-TCDD. We also noticed duction compared to DMSO were obtained to evaluate the ability of that BCZs with one or two halogen substitutions are likely to activate AhR activation (Table 2). AhR more potently than CCZs with corresponding number of halogen Based on the maximal fold induction of the nine PHCZs, the order of substitutions. For instance, both 36-BCZ and 3-BCZ could activate AhR AhR activation ability was 2367-CCZ N 27-BCZ N 1368-CCZ N 136-BCZ (Fig. 1) while their chlor- counterparts, 36-CCZ and 3-CCZ could not N 1368-BCZ N 1-B-36-CCZ N 36-BCZ N 18-B-36-CCZ N 3-BCZ. The AhR ac- (Fig. S5). However, as to congeners with four halogen, the AhR activation tivation potency relative to TCDD was further revealed by estimating potency of BCZs, such as 1368-BCZ was less than CCZs, such as 1368-CCZ, the range of REPs (Table 3). The order of their relative AhR activation suggesting that the number and position of the halogen substitution are potency was 2367-CCZ N 27-BCZ N 136-BCZ N1368-CCZ N 1368-BCZ N also important factors. Interestingly, 1368-CCZ, which has the same num- 1-B-36-CCZ N 36-BCZ N 3-BCZ N 18-B-36-CCZ based on the lowest REP ber of substitute elements but different positions from 2367-CCZ, exhib- values for each PHCZ. Among these PHCZs, 2367-CCZ had the highest ited lower AhR activity than 2367-CCZ with a REP ranging from 1.7E-5 fold induction and REPs ranging from 5.4E-4 to 2.0E-2 (Table 3). The to 5.0E-5 (2367-CCZ: 5.4E-4—2.0E-2). order of the maximal induction fold of the PHCZs (Table 2) was consis- The REP values based on PHCZ-induced CYP1A1/CYP1B1 expression tent with that of the REP values (Table 3) except for 136-BCZ and 3-BCZ. has been reported in MDA-MB-468 human breast cancer cells, which In addition, 3-CCZ and 36-CCZ couldn't activate AhR, and on the con- are generally 10 to 1000 folds higher than that of our study based on trary, they inhibited the luciferase activity upon treatment at nontoxic the DRE-driven luciferase reporter assay (Riddell et al., 2015). The pos- higher concentrations (1E-6 mol/L and 1E-5 mol/L, respectively), sug- sible reason for these differences might be possible involvement of ad- gesting potential antagonizing effects on AhR (Fig. S5). The time course ditional signaling mechanisms in the gene expression compared to study showed that 20—30% and 35—45% inhibitions of luciferase activ- our DRE-driven luciferase reporter assay. In addition to AhR, CYP1A1/ ity were found after 12-h to 48-h treatment with 36-CCZ and 3-CCZ, re- CYP1B1 expression can be mediated by other signaling pathway too, spectively (Fig. S6A and Fig. S6B). These stable inhibitory effects on AhR such as estrogen receptor pathway (Go et al., 2017) and pregnane X re- signaling pathway further support their potential antagonizing effects ceptor (Moriya et al., 2016) which may also contribute to the induction on AhR. of the genes in certain kind of cells. 522 D. Ma et al. / Science of the Total Environment 687 (2019) 516–526

Table 2 Endpoints of PHCZs on the AhR activation in reporter gene assay and their predicted free energies of AhR binding.

b Ligand MOEC EC50 (mol/L) Maximal Predicted binding (mol/L)a fold free energy inductionc (kcal/mol)

TCDD 3.30E−10 2.5E−11 53 −8.1 (1.9E−11–3.3E−11)d 2367-CCZ 1.00E−06 3.4E−09 35 −7.6 (8.1E−10–1.3E−08) 27-BCZ 1.00E−07 9.3E−08 32 −7.3 (2.3E−08–2.1E−07) 1368-CCZ 1.00E−06 5.90E−07 27 −7.1 (3.0E−07–7.4E−07) 136-BCZ 1.00E−06 5.3E−07 25 −7.2 (4.0E−07–6.9E−07) 1368-BCZ 1.00E−06 3.7E−06 21 −6.4 (2.9E−06–4.7E−06) 1-B-36-CCZ 1.00E−06 1.652E−006 15 −6.2 (1.4E−06–1.8E−06) 36-BCZ 1.00E−06 9.50E−07 8 −6.2 (5.9E−07–1.5E−06) 18-B-36-CCZ 1.00E−06 1.50E−07 3 −5.2 (7.1E−08–2.1E−07) 3-BCZ 1.00E−06 1.90E−07 2 −5.9 (9.1E−08–3.8E−07)

a MOEC: maximal observed effect concentration (i.e. the lowest tested concentration causing the maximum effect). b EC50: The half maximum effective concentrations. c Maximal fold induction compared to DMSO. d EC50 values with 95% conﬁdence intervals.

pathway, was determined after treatment with AhR activating PHCZs in CBG 2.8D cells. As shown in Fig. 2, the general induction ability of these PHCZs was consistent with that of the reporter assay. All of the nine PHCZs significantly increased CYP1A1 mRNA levels albeit with different relative induction ability from that of the reporter assay further supporting their action on the AhR signaling pathway. The most potent AhR-activating PHCZ identified here, 2367-CCZ, had the most potent induction effect on CYP1A1 gene expression, which was mostly (95%) re- versed after AhR silencing (Fig. S7). Generally, PHCZs with higher REP values induced CYP1A1 expression in higher degrees (Fig. 2). The order of their induction abilities was 2367-CCZ N 27-BCZ N 1368-BCZ N N Fig. 1. Concentration-dependent luciferase activity induced by TCDD and PHCZs in CBG 1368-CCZ 136-BCZ (Fig. 2). According to this order, 1368-BCZ 2.8D cells. (A) TCDD and PHCZs congener with the strongest AhR activity. (B) PHCZs seemed to have a better induction effect on CYP1A1 compared to the lu- congener with moderate AhR activity. (C) PHCZs congener with the weakest AhR ciferase reporter assay. A relative higher CYP1A1 induction ability of activity. Luciferase activities were determined after 24-hour-treatment with indicated 1368-CCZ has been found in MDA-MB-468 human breast cancer cells chemicals (PHCZ: 1E-12 mol/L to 2E-6 mol/L; TCDD: 1.5E-13 mol/L to 1E-9 mol/L). Data are normalized by solvent control (DMSO) which is set as 1. Values represent means ± (Riddell et al., 2015), which is even higher than that of 2367-CCZ. We SEM, and were obtained from triplicated wells of three independent experiments (n = 3). proposed that the species-or cell-type-specificity of AhR pathway and its interacting pathway may account for the discrepancy (Denison et al., 2011). For those with lower REPs, such as 36-BCZ, 1-B-36-CCZ AhR can be activated by diverse chemicals occurred in environment and 18-B-36-CCZ, the induction effect on CYP1A1 expression was ap- samples (Denison et al., 2011; Nguyen and Bradfield, 2008) and leading parently less than the other PHCZs. The increasing CYP1A1 mRNA to diverse consequences, such as carcinogenesis (Blankenship et al., level might lead to induction of CYP1A1 protein and the enzymatic ac- 2003), alteration of cellular morphology, cell migration, and plasticity tivity as reported by Riddell et al., 2015 for certain PHCZs. However, (Bui et al., 2009; Diry et al., 2006; Tomkiewicz et al., 2013). PHCZs rep- the toxicity related consequences of the CYP1A1 gene induction needs resent a new class of dioxin-like compounds which might affect a bat- further investigation. tery of effective genes and induce toxic effects through AhR signaling The effect on the expression of responsive gene of AhR signaling pathway. Due to the relatively high levels of PHCZs measured in envi- pathway might not only rely on the binding affinity of AhR ligand but ronment samples, for example, up to 440 ng/g in the sediment of Lake also the ligand-dependent structural and functional specificity of the ac- Tai in China (Wu et al., 2016), the potential adverse effect of PHCZs tivated AhR protein, and its related protein-or DNA-binding-complex, should be taken into consideration albeit with the low REPs. which has been proposed for structurally diverse ligands of AhR (Gouédard et al., 2004b; Ma, 2001; Zhao et al., 2010). Therefore, in the 3.2. PHCZs increase CYP1A1 gene expression following part of the study, we employed computational analysis and a ligand-selective AhR antagonist, CH223191 (Zhao et al., 2010)tochar- In order to validate the downstream effect upon activation of AhR, acterize the potential interaction between AhR and the AhR-activating mRNA expression of CYP1A1, a typical responsive gene of AhR signaling PHCZs identified in this study. D. Ma et al. / Science of the Total Environment 687 (2019) 516–526 523

Table 3 Maximal fold of induction of TCDD (positive control, PC) and Relative Effect Potencies of PHCZs on the AhR activation in reporter gene assay.

Ligand PC10 PC20 PC50 PC80 REP range (mol/L) (mol/L) (mol/L) (mol/L)

TCDD 3.4E−12 8.4E−12 2.5E−11 7.5E−11 1.0–1.0 (5.35)a (10.69)a (26.74)a (42.78)a 2367-CCZ 1.7E−10 8.2E−10 4.7E−08 NE 5.4E−4–2.0E−2 27-BCZ 5.8E−09 2.7E−08 4.5E−07 NE 5.6E−5–6.0E−4 136-BCZ 7.5E−08 2.3E−07 1.1E−06 2.7E−06 2.3E−5–4.6E−5 1368-CCZ 7.0E−08 2.6E−07 1.5E−06 3.5E−06 1.7E−5–5.0E−5 1368-BCZ 2.0E−07 4.1E−07 1.7E−06 NE 1.5E−5–2.0E−5 1-B-36-CCZ 3.1E−07 8.3E−07 1.4E−05 NE 1.9E−6–1.1E−5 36-BCZ 6.1E−07 5.9E−06 NE NE 1.4E−6–5.7E−6 3-BCZ 8.4E−07 NE NE NE NE 18-B-36-CCZ NE NE NE NE NE

PC10,PC20,PC50,PC80: the concentration of compound reaching 10%, 20%, 50% or 80% of the Fig. 2. Induction of CYP1A1 by PHCZs. CBG 2.8D cells were treated with indicated PHCZs at maximal response of TCDD (positive control (PC). The relative effect potency (REP) values 1E-6 mol/L for 24 h. CYP1A1 mRNA levels were determined by qRT-PCR analysis as “ ” were calculated as TCDD(PCx)/PHCZ(PCx). No range of REP values was presented when mentioned in Materials and Methods . Beta-actin served as an internal control for ﬁ only the REP PC10 could be calculated. NE: Not estimated. quanti cation. Values represent means ± SEM, and were obtained from triplicated wells a 10%, 20%, 50% or 80% of maximal TCDD response (fold of induction normalize by of three independent experiments (n = 3). *p b 0.05, **p b 0.01, signiﬁcant difference DMSO). from the solvent control.

complex stability provided by nonpolar interactions. Specifically, 3.3. In silico study on AhR-PHCZ interaction 2367-CCZ, the perfect PHCZs counterpart of TCDD, had the highest predicted binding potential but still significantly lower than TCDD (Fig. 3B). The interaction between AhR and the PHCZs can be established by One of the distinguished changes in the binding mode of 2367-CCZ rel- employing molecular docking on the obtained LBD model since docking ative to that of TCDD was the missing π-π interactions between Phe38 studies can create a better picture of such interaction and their role in and the ligand. One phenyl ring in TCDD established π-σ interactions the transcriptional activation process. Homology modeling based on tem- with Leu67 instead. In addition to the skeleton effect, the halogenated plates has been performed to get the modeled mAhR-LBD since there is no type, position, and degree on the two phenyl rings also played an impor- three-dimensional structure of mAhR-LBD in the RCSB Protein Data Bank tant role in the regulating the binding affinity. The large volume of bro- (Fig. S1). The resulting QMEAN Z-score is a measure of the ‘degree of na- mine atom on the phenyl ring was not favorable for the receptor-ligand tiveness’ of the structure feature observed in our model and thus indicates recognition, and the meta-halogenation was more beneficial to the li- whether the model is of comparable quality to the experimental struc- gand binding than the ortho- arrangement due to steric effect. The cal- tures. The closer the value of Z-score is to zero, the better the model. culated binding affinity was in the similar order, that is TCDD N2367- The Z-score of the individual tern ‘torsion’ implied significant deviation CCZ N 27-BCZ N 136-BCZ ≅ 1368-CCZ N 1368-BCZ N 1-B-36-CCZ ≅ 36- the model exhibited from the expected ‘native’ behavior and might BCZ N 3-BCZ N18-B-36-CCZ, as the experimental AhR activation potency come from back-bone geometry, while the overall QMEAN4Z-score of based on the lowest REP values for each PHCZ (Table 2 and Table 3). −2.86 indicated a satisfied model quality for the subsequent study Thus, the present experimental and in silico data on AhR activation pro- (Fig. S3A). The model quality was also verified by the expected similarity vide direct evidence of AhR-PHCZ interaction, which might be upstream to the native structure per residue (Fig. S3B). Clearly, the majority of res- molecular initiation event for the reported CYP1A1/CYP1B1 induction idues showed a score above 0.6, presenting high model quality even with in vitro (Riddell et al., 2015) and dioxin-like cardiotoxicities in vivo low query coverage. Furthermore, the residues with a score below 0.6 are (Fang et al., 2016). far away from the pocket. In addition, the Ramachandran plot in Fig. S4 validated the stereo-chemical structure of the constructed model since 3.4. Differential antagonizing effects of CH223191 on PHCZ-induced AhR 93.1% residues in the LBD appeared in the favored regions. Only few res- activity idues (2.9%) located far away from the binding pocket. The receptor-ligand interaction of TCDD, a typical AhR agonist, was CH223191 is known to preferentially inhibit the ability of certain dominated by van der Waals contacts provided by key residues Leu22, classes of halogenated aromatic hydrocarbons, such as TCDD, but not Tyr24, Leu29, Phe38, Ile39, Cys47, His51, Met62 and Leu67 (The others, such as some polycyclic aromatic hydrocarbons, to activate the amino acid code of the mAhR-LBD starts from 1). There were only few AhR and its signal transduction (Sun-Hee et al., 2006). Whether additional interactions that stabilized AhR-TCDD complex, including CH223191 can antagonize the PHCZ-induced AhR-dependent gene ex- hydrogen bonds between Cys14 side chains and the chlorine atoms in pression was examined by using the luciferase reporter assay. With TCDD, and a π-π interaction between the phenyl ring in Phe38 and pre-treatment of CH223191, we found that TCDD-induced luciferase ac- the TCDD aromatic moiety (Fig. 3A). All the nine PHCZs of AhR activity tivity was inhibited by 90% at highest concentration (CH223191: 1E- could be docked into the same pocket of TCDD with different predicted 5mol/L)(Fig. 4A). The PHCZs being able to activate AhR can be classified free energies of AhR binding, which was attributed to the high structural into two categories based on the antagonizing effect of CH223191: similarity between TCDD and PHCZs. PHCZs replicates the tricyclic (1) CH223191 had the least and no antagonizing effect (b 20% at 1E- structure of TCDD, consisting of two six-membered benzene rings 5 mol/L) on the PHCZs, including 27-BCZ and 2367-CCZ, respectively, fused on either side of a heterocyclic fragment. The only difference be- which were two most potent AhR agonists among the test PHCZs tween the two skeletons is whether the fused heterocyclic substructure (Fig. 4A); (2) CH223191 had the moderate to strong antagonizing effect is pyran or pyrole ring. Therefore, the 9 PHCZs established nonpolar in- (approximately 75—50% at 1E-5 mol/L) on them, including the other teractions, such as van der Waals contacts, π-σ and π-π interactions, seven PHCZs (Fig. 4B), which had weak to moderate AhR activation abil- through the residues like Gly35, Phe38 and Leu67 to stabilize the AhR- ity in terms of the REP values (Table 3, Fig. 4B). Interestingly, treatment PHCZs complexes. Nevertheless, the decrease in the expansion extent of CH223191 alone slightly inhibited the AhR activity compared to sol- of the rigid skeleton, caused by the shrunk size of the heterocyclic moi- vent control suggesting the presence of endogenous AhR activation in eties in PHCZs relative to that in TCDD, dramatically decreased the CBG 2.8D cells (Fig. S8). 524 D. Ma et al. / Science of the Total Environment 687 (2019) 516–526

Fig. 3. AhR Interaction with TCDD and the PHCZs. Docking analysis was implemented as mentioned in “Materials and Methods”.ThemAhR-LBD binding modes with TCDD (A), 2367-CCZ (B) and 2,7-BCZ (C), and the two distinguished binding poses of 2367-CCZ (green) and 2,7-BCZ (green) (D), and other 7 PHCZs (green) (E) in comparison with that of TCDD (yellow) are shown.

The in silico analysis also supported the above categorization of binding features for TCDD and the PHCZs (Fig. 3), that of TCDD pose, the antagonizing effect of CH223191 on the nine PHCZs. In fact, the way of 2367-CCZ and 27-BCZ, the mode symbolized by other there were two distinguished poses adopted by the TCDD and PHCZs. The different hydrogen bonding effect and π-π interaction PHCZs in the AhR-LBD (Fig. 3). One was illustrated by 2367-CCZ can cause differentiated transcriptional activation potential. The and 27-BCZ which adopt the binding modes obviously differentiated pose difference among the ligands in a receptor may lead to the from that observed in the TCDD-AhR complex, while the other was change in the protein surface structure and thus affects subsequent symbolized by TCDD and the other seven AhR agonists. The existence transcriptional activation process, and the similar situation of of -NH- moiety in 2367-CCZ and 27-BCZ made the rigid tricyclic bisphenol AF has already been reported for estrogen receptor with plane moving towards Thr3, which was driven by hydrogen bonding solid proof from its co-crystallized structure (Vanessa et al., 2012). (Fig. 3BandFig. 3C), and the two ligands were rotated by about 10 degree clockwise with respect to the TCDD molecular plane by Declaration of Competing Interest their interactions with the pocket (Fig. 3D). On the contrary, there were no hydrogen bonds involving the O atoms in TCDD (Fig. 3A). The authors declare that they have no conﬂict of interest. No hydrogen bonds were observed for the –NH- moiety in other PHCZs as well (Fig. S9). Moreover, the other PHCZs were rotated Acknowledgments counterclockwise with respect to the TCDD molecular plane when bound to AhR (Fig. 3E). Clearly demonstrated by the orientation of This work was supported by the Strategic Priority Research Program the ring plane in the binding pocket, there are three kinds of different of the Chinese Academy of Sciences (Grant Nos. XDB14030401 and D. Ma et al. / Science of the Total Environment 687 (2019) 516–526 525

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