Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons

Environmental Research 151 (2016) 106–114

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Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons Konrad A. Szychowski a,b, Agnieszka Wnuk c, Małgorzata Kajta c, Anna K. Wójtowicz b,n a Department of Public Health, Dietetics and Lifestyle Disorders, Faculty of Medicine, University of Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225 Rzeszow, Poland b Department of Animal Biotechnology, Animal Sciences Faculty, University of Agriculture, Redzina 1B, 30-248 Krakow, Poland c Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Poland

art ic l e i nf o

a b s t r a c t

Article history: Received 19 January 2016 Received in revised form 20 June 2016 Accepted 13 July 2016

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitizing products, such as soaps, toothpastes, and hair products. A number of studies have revealed the presence of TCS in human tissues, such as fat, liver and brain, in addition to blood and breast milk. The aim of the present study was to investigate the impact of TCS on AhR and Cyp1a1/Cyp1b1 signaling in mouse neocortical neurons in primary cultures. In addition to the use of selective ligands and siRNAs, expression levels of mRNA and proteins as well as caspase-3 activity, reactive oxygen species (ROS) formation, and lactate dehydrogenase (LDH) release have been measured. We also studied the involvement of the AhR in TCS-induced LDH release and caspase-3 activation as well as the effect of TCS on ROS generation. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine, and the neurons were exposed to 1 and 10 mM TCS. Our experiments showed that the expression of AhR and Cyp1a1 mRNA decreased in cells exposed to 10 mM TCS for 3 or 6 h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3 h of exposure to TCS, but after 6 h, the mRNA expression of Cyp1b1 was decreased. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and pro-apoptotic properties of TCS were decreased. The decrease in Cyp1a1 mRNA and protein expression levels accompanied by a decrease in its activity. The stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS, whereas the addition of AhR antagonist (αNF) reversed the inhibitory effects of TCS. In our experiments, TCS diminished Cyp1b1 mRNA and enhanced its protein expression. In case of Cyp1a1 we observed paradoxical effect of TCS action, which caused the decrease in activity and protein expression of Cyp1a1 and the increase in protein level of AhR. Therefore, we determined the effects of TCS on the production of ROS. Our results revealed that TCS increased the production of ROS and that this effect of TCS was reversed by 10 mM Nacetyl-L-cysteine (NAC), the ROS scavenger. To confirm an involvement of ROS in TCS-induced neurotoxicity we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and NAC. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6 h, respectively. We postulate that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCS-induced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS. & 2016 Elsevier Inc. All rights reserved.

Keywords: Triclosan AhR Cyp1a1 Cyp1b1 Apoptosis Neuron

1. Introduction Triclosan

n

(IUPAC

name:

5-Chloro-2-(2,4-dichlorophenoxy)

Corresponding author. E-mail address: [email protected] (A.K. Wójtowicz).

http://dx.doi.org/10.1016/j.envres.2016.07.019 0013-9351/& 2016 Elsevier Inc. All rights reserved.

phenol; CAS number: 3380-34-5) (TCS), also known as Irgasan, Microban or Cloxifenolum, is widely used as an antibacterial agent. TCS is present in numerous personal care and sanitizing products, and household items (Bhargava and Leonard, 1996; Perencevich et al., 2001; Møretrø et al., 2011; McArthur et al., 2012; Liu et al., 2015). Due to its solubility properties, TCS has been detected in water and sediments (Zhang et al., 2013; Pintado-Herrera et al.,

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2014). TCS exhibits lipophilic properties and also can easily pass through biological barriers (Pannu et al., 2012). A number of studies have revealed the presence of TCS in human tissues, such as the blood (Allmyr et al., 2006, 2008), adipose, liver, and brain (Geens et al., 2012; Wang et al., 2015), in addition to breast milk (Allmyr et al., 2006; Toms et al., 2011) and urine (Kim et al., 2011; Mortensen et al., 2014). Bagley and Lin (2000) reported that humans are exposed to a chronic systemic circulation of 14–21 ng/mL TCS in the blood (E 48.61–72.91 nM). However, Mustafa et al (2003) demonstrated that TCS can be distributed throughout the cytoplasm and nuclei of cells in vitro, suggesting that a certain amount of TCS can accumulate in the human body at higher levels than what has been reported in the blood. Although TCS has been detected in fish (13–88 ng/g E47.14–319.12 nM) and human (0.23 ng/g E0.83 nM) brains (Geens et al., 2012; Tanoue et al., 2014) and can disrupt the function of zebrafish (Danio rerio) motor neurons (Muth-Köhne et al., 2012), only two papers has examined the mechanism of action of TCS in neuronal cells (Szychowski et al., 2015; Park et al., 2016). The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes that are primarily activated by xenobiotics, especially dioxin (Eriksson and Talts, 2000). It is well known that TCS has a similar chemical structure to dioxin and, in the presence of sunlight, can be transformed into as many as four dioxin compounds, such as 2,8-DCDD, 2,3,7-TCDD, 1,2,8-TriCDD, and 1,2,3,8-TCDD (Buth et al., 2009). However, a recent paper has shown that TCS can also be transformed to 2,8-DCDD without sun light at room temperature and under near dry conditions (Ding et al., 2015). The activation of the AhR results in the transcription of cytochrome P450 enzymes (CYP), such as Cyp1a1 and Cyp1b1 (Guengerich et al., 2003). Cyp1a1 and Cyp1b1 are involved in the metabolism of many endogenous compounds, such as cholesterol, steroid hormones, and environmental xenobiotics (Kawajiri and Fujii-Kuriyama, 2007). Both Cyp1a1 and Cyp1b1 are 17β-estradiol (E2) hydroxylases. Cyp1a1 has activity at the C-2, C-6α and C-15α positions of E2, and CYP1B1 has its primary activity at C-4 with a 5-fold lower activity at C-2 (Spink et al., 1992; Hayes et al., 1996). Cyp1a1 and Cyp1b1 are known to catalyze the formation of mutagenic intermediates from a number of polycyclic aromatic hydrocarbons (PAH), including several that are potent mammary gland carcinogens in rodents (Shimada et al., 1992, 1996). Cyp1b1 appears to be more active than Cyp1a1 in the conversion of a number of PAHs to genotoxic intermediates (Shimada et al., 1996; Spink et al., 1998). Cyp1b1 also catalyzes the metabolic activation of several aryl amines and heterocyclic amines, and seams to play more crucial role in carcinogenesis (Shimada et al., 1996; Spink et al., 1998). Interestingly, it has been well shown that some xenobiotics can inhibit the activity of many metabolizing enzymes, such as Cyp1a1, Cyp1b1 and UDP-glucuronosyltransferase (Pollock et al., 2014). It has been demonstrated that AhR is also involved in the regulation of the expression of genes that control the growth and differentiation of neurons and in their metabolism and adaptation (Barouki et al., 2007; Lindsey and Papoutsakis, 2012). In primary cell cultures from the neocortex, hippocampus and cerebellum, apoptosis has been observed that is dependent on the activation of the AhR (Kajta et al., 2009, 2014). To date, it has been shown that TCS can interact with the AhR in a rat hepatoma cell line (H4L1.1c4) (Ahn et al., 2008). Despite the fact that TCS activates the AhR, there have been no additional studies of the role of this receptor in the mechanism of action of TCS. Interestingly, data related to the AhR-dependent activation of Cyp1a1 and Cyp1b1 by TCS are inconsistent (Hanioka et al., 1996; Paul et al., 2009; Liang et al., 2013; Ku et al., 2014). The aim of the present study was to understand the role of AhR-signaling and related Cyp1a1/Cyp1b1 proteins and also the

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ROS formation in TCS neurotoxicity in mouse neocortical neurons in primary cultures.

2. Materials and methods 2.1. Reagents Neurobasal medium without phenol red, B27-AO supplement and the TaqMan probes corresponding to specific genes encoding β-actin (Mm00607939_s1), AhR (Mm01291777_m1), Cyp1a1 (Mm00487218_m1), and Cyp1b1 (Mm00487229_m1) were purchased from Life Technologies (Forest City, CA, USA). Trypsin, charcoal/dextran-treated fetal bovine serum (FBS), penicillin, streptomycin, staurosporine, triclosan (Irgasan), N-acetyl-L-cysteine (NAC), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The LDH-based cytotoxicity detection kit was purchased from Roche Applied Science (Mannheim, Germany). PBS was purchased from BIOMED (Lublin, Poland). Caspase-3 substrate was purchased from Calbiochem (Merck Corporation, Darmstadt, Germany). The INTERFERin siRNA transfection reagent was purchased from Polyplus-transfection (Illkirch, France). The AhR siRNA (sc-29655) and anti-AhR (sc-8088), antiCyp1a1 (sc-9828) and anti-Cyp1b1 (sc-32882) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). TCS and other reagents were dissolved in DMSO. Controls in all experiments were DMSO-treated cells. Results were compared to a vehicle control in all experiments. DMSO has been used in a concentration of 0.01%. In this concentration DMSO had no significant effect on caspase-3 activity, LDH release, and ROS formation, but affected EROD assay. However, to analyze properly the effects of TCS on CYP1A1 activity, control cultures were treated with 0.01% DMSO which was the solvent of TCS in TCS-treated cultures. 2.2. Primary cultures of neocortical neurons The experiments were performed on primary cultures of mouse neocortical neurons. These cultures were prepared from the fetuses of pregnant female Swiss mice as previously described by Brewer (1995) and modified by Szychowski et al. (2015). Brain tissues were collected from mouse embryos on day 15 or 16 of gestation. Pregnant females were anesthetized with CO2 vapor and killed by cervical dislocation. The animal care protocols were in accordance with official governmental guidelines, and all efforts were made to minimize the number of animals used and their suffering. All procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Bioethics Commission (No. 46/2014) in compliance with Polish law. The brains were removed from the fetuses, and the cortical tissues were dissected. The dissected tissue was minced into small pieces and then gently digested with trypsin. Then, the cells were centrifuged, and the pellet was suspended in phenol red-free Neurobasal medium supplemented with 5% charcoal/dextran-treated FBS. The cells were plated onto poly-L-ornithine-coated (0.01 mg/mL) multi-well plates. After 2 days, the culture medium was exchanged with Neurobasal medium supplemented with B27-AO (2 μL/mL), glutamine (2 mM), 10 U/mL penicillin, and 0.01 mg/mL streptomycin, which is recommended for primary neuronal cultures (Brewer, 1995; Kajta et al., 2005). For the experiments, the cells were cultured at a density 1.8
105 cells/cm2. This procedure typically yields cultures that contain approximately 90% neurons and 10% astrocytes (Kajta et al., 2004). The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and were

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cultivated for 7 days prior to the experiment. Afterwards, the culture medium was changed prior to treating the cultures with the compounds selected for this study. 2.3. siRNA gene silencing procedure AhR siRNA was used to inhibit AhR expression in mouse neocortical neurons using a modification of a previously described method (Kajta et al., 2014). The siRNA was applied for 7 h at a final concentration of 50 nM in antibiotic-free medium containing the siRNA transfection reagent INTERFERin. For the experiments, the cells were plated on 96-well plates. After transfection, the culture medium was changed, and the neurons were cultured for 12 h prior to starting the experiment. Vehicle controls included positive siRNA and negative siRNA containing a scrambled sequence that did not lead to the specific degradation of any known cellular mRNA. The effectiveness of AhR mRNA silencing with the use of 50 nM specific siRNA was verified by the measurements of mRNA and protein levels. Knockdown of AhR was estimated at 26% of the vehicle control mRNA, and at 35% of the vehicle control protein level as previously described (Kajta et al., 2014; Rzemieniec et al., 2015). After 12 h, cells were treated with 1 or 10 μM TCS, 10 mM AhR agonist beta-naphthoflavone (βNF), or 1 mM AhR antagonist alpha-naphthoflavone (αNF) for 24 h and LDH and caspase-3 activities were determined. 2.3.1. LDH cytotoxicity assay The cytotoxicity detection kit is a colorimetric assay for the quantification of cell death and cell lysis based on the release of LDH from the cytosol of damaged cells into the surrounding medium (Koh and Choi, 1987). An increase in the amount of dead or plasma membrane-damaged cells results in an increase in LDH activity in the culture medium. Following 24 h of treatment 100 μL of the culture supernatants were collected to determine LDH levels, and the cells were frozen at  80 °C to measure caspase-3 activity. For cytotoxicity measurements, the reaction was stopped after 30 min by adding 1 N HCl, and the absorbance at a wavelength of 490 nm was measured using an ELISA microplate reader manufactured by Bio-Tek Instruments Inc. The results are expressed as the mean percentages 7 SEM relative to the vehicle control from eight separate samples, and the samples were measured in quadruplicate. 2.3.2. Caspase-3 activity Caspase-3 activity was used as a marker of cell apoptosis and was assessed according to Nicholson et al. (1995). After thawing from  80 °C storage, the neurons were lysed using lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM DTT) at 10 °C for 10 min. The lysates were incubated in the caspase-3 substrate Ac-DEVD-pNA at 37 °C. Cells treated with 1 μM staurosporine were used as a positive control (results not shown). After 30 min, the absorbance of the lysates at 405 nm was measured using a microplate reader (Bio-Tek ELx800). The amount of colorimetric product was continuously monitored for 120 min. The data were analyzed using KCJunior software (BioTek Instruments Inc. and were normalized to the absorbance of the vehicle-treated cells. The results are expressed as the mean percentage 7 SEM relative to the vehicle control from eight separate samples, and the samples were measured in quadruplicate. 2.4. Measurement of TCS-stimulated reactive oxygen species (ROS) production The fluorogenic dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular reactive oxygen species (ROS). After diffusing into the cell, H2DCFDA is de-acetylated

by cellular esterases into a non-fluorescent compound that is subsequently oxidized by ROS into 2′,7′-dichlorofluorescein (DCF) (Gomes et al., 2005). To determine the ability of TCS to induce ROS production in neocortical neurons, we applied 5 μM H2DCFDA. For the ROS measurement, the cells were plated on black-sided, clearbottomed 96-well plates and exposed to 1 or 10 mM TCS for 3 or 6 h. The cells were incubated in H2DCFDA in serum-free and phenol red-free Neurobasal medium for 45 min prior to TCS treatment. After 3 or 6 h of incubation of the cells with TCS (5% CO2 at 37 °C), the culture medium was replaced with fresh Neurobasal medium to remove residual extracellular DCF and TCS to reduce the fluorescence background. The addition of 10 mM NAC ROS scavenger was used to reduce the effect of TCS-stimulated ROS production. Cells treated with 55 mM tert-butyl hydrogen peroxide (TBHP) were used as a positive control (results not shown). The interaction between TCS and H2DCFDA was tested under cell-free conditions prior to the experiments (results not shown) based on concerns about the H2DCFDA assay previously described by Szychowski and Wójtowicz (2016). DCF fluorescence was detected using a microplate reader (Bio-Tek FLx800) at maximum excitation and emission spectra of 485 nm and 535 nm, respectively. The data were analyzed using KCJunior software (BioTek Instruments Inc.) and was normalized to the fluorescence in the vehicle-treated cells (% of control). The means 7 SEM from eight separate samples were calculated from four independent experiments. 2.5. EROD assay We estimated the activity of the Cyp1a1 enzyme using the fluorometric ethoxyresorufin-O-deethylase (EROD) assay. The fluorescent EROD assay for Cyp1a1 activity was performed according to the method of Kennedy et al. (1993). The total protein concentration in each well was simultaneously measured using fluorescamine according to the method of Kennedy and Jones (1994). For the EROD assay, neurons were seeded on poly-L-ornithine-coated 6-well plates and were initially cultured for 7 days. The measurement of Cyp1a1 activity was performed following 48 h of exposure to 10 mM TCS and co-treatment with 10 mM TCS and 10 mM βNF or 1 mM αNF (cells were exposed βNF or αNF 1 h before the experiment). Controls with βNF or αNF were included in the experiment to determine the effects of those compounds. To perform the EROD assays, the lysed cells were transferred to multiwell plates, and the fluorescent product, resorufin, and the total amount of protein were quantified within the same wells using a fluorescence plate reader (Bio-Tek Instruments Inc.). The ethoxyresorufin metabolite, resorufin, was measured using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The protein concentration was measured using fluorescamine at an excitation wavelength of 400 nm and an emission wavelength of 460 nm. 2.6. Real-time PCR analysis of mRNAs specific to genes encoding the AhR, Cyp1a1 and Cyp1b1 For the real-time PCR assay, neurons were seeded on poly-Lornithine-coated 6-well plates and initially cultured for 7 days. After 3 or 6 h of exposure to 10 mM TCS, and in co-treatment TCS with 10 μM NAC, samples were collected, and total RNA was extracted from neocortical neurons using a Qiagen RNeasy mini kit according to the manufacturer’s protocol based a previously described method (Kajta et al., 2014). The quality and quantity of the RNA were determined spectrophotometrically at 260 nm and 280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). Twostep real-time RT-PCR was conducted with both the reverse transcription (RT) reaction and the quantitative polymerase chain

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reaction (qPCR) run using the CFX96 Real Time System (BioRad, USA). The RT reaction was performed at a final volume of 20 μL with 300 ng of RNA (as a cDNA template) using the cDNA reverse transcription kit according to the manufacturer’s protocol. The products from the RT reaction were amplified using the TaqMan Gene Expression Master Mix (Life Technologies Applied Biosystems, USA) kit with TaqMan probes as primers for the specific genes encoding AhR, β-actin, Cyp1a1 and Cyp1b1. Amplification was carried out in a total volume of 20 μL containing 1
TaqMan Gene Expression Master Mix and 1 μL of RT product, which was used as the PCR template. Standard qPCR procedures were performed as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was set during the exponential phase, and the Δ Ct method was used for data analysis. β-actin was used as the reference gene. 2.7. Western blot analysis For the Western blot assay, neurons were seeded on poly-Lornithine-coated 6-well plates and were initially cultured for 7 days. After 1, 3, 6, 24 or 48 h of exposure to 10 mM TCS, Western blot samples were collected and AhR, Cyp1a1, and Cyp1b1 protein expression levels were measured. For immunoblotting, the cells were lysed in 100 μL of ice-cold lysis buffer containing 100 mM NaCl, 50 mM Tris HCl (pH 7.5), 0.5% Na-deoxycholate, 0.5% Nonidet NP-40% and 0.5% SDS. Then, the lysates were sonicated and clarified by centrifugation at 15,000
g at 4 °C for 20 min, and the supernatant was collected and stored at  80 °C until analysis. The protein concentrations in the supernatants were determined using the Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as the standard. From the whole cell lysate, 40 mg of total protein were reconstituted in the appropriate amount of sample buffer, which consisted of 125 mM Tris (pH 6.8), 4% SDS, 25% glycerol, 4 mM EDTA, 20 mM DTT and 0.01% bromophenol blue. Samples were separated by 7.5% SDS-polyacrylamide gel electrophoresis in a Bio-Rad Mini-Protean II Electrophoresis Cell, and the proteins were then transferred to nitrocellulose membranes using a Bio-Rad Mini Trans-Blot apparatus. Following the transfer, the membranes were washed, and nonspecific binding sites were blocked with 5% dried milk and 0.2% Tween 20 in 0.02 M TBS for 2 h. Then, the membranes were incubated overnight with antiAhR, anti-Cyp1a1, and anti-Cyp1b1 antibodies diluted at 1:200 in TBS/Tween at 4 °C. Following incubation with the primary antibodies, the membranes were washed with TBS and 0.02% Tween 20 and then incubated for 2 h with horseradish peroxidase-conjugated secondary antibodies diluted to 1:1000 in TBS/Tween. To control for the amount of protein that was loaded onto the gel, we used an anti-GAPDH antibody diluted at 1:1000 in TBS/Tween (secondary antibody diluted at 1:5000 in TBS/Tween). Signals were detected by chemiluminescence (ECL) using a western blotting luminol reagent and visualized with the use of a PhosphorImager FujiLas 4000.

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Statistica 10 software. ***P o0.001,**Po 0.01, and *P o0.05 vs. the vehicle control cultures.

3. Results 3.1. Effects of AhR on TCS toxicity 3.1.1. The involvement of the AhR in TCS-induced LDH release Following 24 h of exposure to 1 or 10 mM concentrations of TCS, the neurons transfected with negative siRNA showed an increase in LDH release compared with the vehicle control in only the 10 mM condition (increase by 99.67%). Following transfection with AhR siRNA, we observed a decrease in LDH release compared with the neurons transfected with negative siRNA (decreased by 34.89%) (Fig. 1A). 3.1.2. The involvement of the AhR in TCS-induced caspase-3 activity Following 24 h of exposure to 1 and 10 mM TCS, the neurons transfected with negative siRNA showed enhanced caspase-3 activity compared with the vehicle controls in both 1 and 10 mM TCS conditions (increase of 23.57% and 141.42%, respectively). In the cells transfected with AhR siRNA, 1 mM TCS did not activate caspase-3. In these cells, 10 mM TCS retained its ability to activate caspase-3, but the level of caspase-3 activity was reduced by 42% compared with the cells transfected negative siRNA (Fig. 1B). 3.2. Effects of TCS on the expression of AhR, Cyp1a1 and Cyp1b1 mRNA Following 3 h of exposure to 10 mM TCS, the neocortical neurons showed a decrease in their expression of AhR and Cyp1a1 mRNA compared with the vehicle control (decrease of 27.18% and 22.00%, respectively). After 3 h of simultaneous administration of

2.8. Statistical analysis The data are presented as the means 7 SEM from four independent experiments. Each treatment was repeated eight times (N ¼ 8) and measured in quadruplicate; thus, the total number of replicates was 32. The average of the quadruplicate samples was used for the statistical analyses. Statistical analysis was performed on the original results. Considering the different data from the measurement of fluorescence or absorbance, the results are presented as percent of controls. The data were analyzed via one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison procedure. Statistical data analysis was performed using

Fig. 1. The effects of 1 and 10 mM TCS, 10 mM βNF or 1 mM αNF on the release of LDH (A) and caspase-3 activity (B) on negative siRNA- and AhR siRNA-transfected cells after 24 h of exposure. Data are expressed as the means 7 SEM of four independent experiments, each of which is comprised of eight replicates per treatment group. ***Po 0.001 vs. the vehicle control, ###Po 0.001 vs. the cells transfected with negative-siRNA.

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3.3. TCS-stimulated ROS production Following 3 h of exposure to 1 and 10 mM TCS, both concentrations increased ROS production in the neurons compared with the controls (increases of 19.31% and 49.32%, respectively). The presence of 10 mM NAC reduced the production of ROS induced by TCS back to control levels (Fig. 3A). Following 6 h of exposure to 1 and 10 mM TCS, both concentrations increased ROS production in the neurons compared with the controls (increases of 20.47% and 62.46%, respectively). The presence of 10 mM NAC decreased the production of ROS induced by TCS back to control levels (Fig. 3B). 3.4. Effects of TCS on the expression of AhR, Cyp1a1 and Cyp1b1 protein

Fig. 2. The effect of 10 mM TCS, 10 mM NAC and TCS with NAC on the mRNA expression of AhR, Cyp1a1 and Cyp1b1 after 3 h (A) and 6 h (B) of exposure. mRNA expression was normalized to β-actin. The data are expressed as the means 7 SEM of four independent experiments, each of which consisted of eight replicates per treatment group. *P o 0.05; ***Po 0.001 vs. the vehicle control. #Po 0.05; ##P o0.01; ###Po 0.001 vs. the TCS treated cells.

TCS and NAC ROS scavenger, the expression of AhR mRNA was decreased by 40.67%, compared with the controls; however, Cyp1a1 and Cyp1b1 expression was increased by 31.83% and 90.54% respectively (Fig. 2A). Following 6 h of exposure to 10 mM TCS, we observed a decrease in the expression of AhR, Cyp1a1, and Cyp1b1 mRNA compared with the vehicle control (decrease of 34.45%, 43.19% and 48.06%, respectively). After 6 h simultaneous administration of TCS and NAC, we observed that the presence of NAC enhanced by 73.15% the expression of AhR mRNA compared with the controls but decreased the expression of Cyp1b1 by 71.03% (Fig. 2B).

Immunoblot analyses quantified by densitometry demonstrated that in the neurons treated with 10 μM TCS for 24 or 48 h, the level of the AhR protein was increased compared with the vehicle control cells by 67.82% and 81.65%, respectively. The decrease in Cyp1a1 protein expression was observed after 1, 3, 6, 24 and 48 h of exposure to 10 μM TCS (compared with the control, 27.02%, 56.84%, 58,48%, 83.62% and 88.79%, respectively). Cyp1b1 protein expression first decreased after 3 h (by 29.95% compared with the control) and then suddenly peaked at 24 and 48 h (increased by 81.71% and 427.78%, respectively) (Fig. 4). 3.5. Effect of AhR agonist and antagonist on Cyp1a1 activity and protein expression The addition of 10 mM TCS after 48 h significantly decreased the activity of Cyp1a1. This effect was partially reversed by βNF and completely reversed by αNF. Additionally, the AhR agonist βNF produced an increase in the activity of Cyp1a1, whereas the AhR antagonist αNF did not affect the activity of this enzyme (Fig. 5A). The addition of 10 mM TCS after 48 h significantly decreased the protein expression of Cyp1a1 by 29.34% compared to control. After co-administration of TCS, and βNF or αNF protein expression of Cyp1a1 decrease by 26.17% and 35.02% respectively compared to control (Fig. 5B, C).

4. Discussion Our study is the first to examine the impact of TCS on the

Fig. 3. The effect of 1 and 10 mM TCS on ROS production after 3 (A) and 6 (B) h. The addition of 10 mM NAC reduced the effect of TCS. The data are expressed as the means 7 SEM of four independent experiments, each of which consisted of eight replicates per treatment group. **P o 0.01; ***Po 0.001 vs. the vehicle control. #Po 0.05; ##P o0.01; ###P o 0.001 vs. the cells without NAC.

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Fig. 5. Effects of 10 mM TCS, 10 mM βNF and 1 mM αNF alone and co-treatment of 10 mM TCS, with 10 mM βNF or 1 mM αNF on Cyp1a1 activity in cultured mouse neurons following 48 h of exposure. The data are expressed as the means 7 SEM of four independent experiments, each of which consisted of eight replicates per treatment group (A). Representative western blot of Cyp1a1 protein levels from neocortical neurons treated with 10 mM TCS, 10 mM βNF and 1 mM αNF alone and cotreatment of 10 mM TCS, with 10 mM βNF or 1 mM αNF after 48 h (B). Protein bands were quantified by densitometry. The results are shown as the percentage of Cyp1a1 proteins relative to the control. Each column represents the mean 7 SEM of three independent experiments (C). **Po 0.01, ***Po 0.001 vs. the vehicle control; #P o0.05, ###Po 0.001 vs. the cells with TCS alone.

Fig. 4. Representative western blot of AhR, Cyp1a1 and Cyp1b1 protein levels from neocortical neurons treated with 10 μM TCS after 1, 3, 6, 24 or 48 h (A). Protein bands were quantified by densitometry. The results are shown as the percentage of AhR, Cyp1a1 and Cyp1b1 proteins relative to the control. Each column represents the mean 7 SEM of three independent experiments (B-D). The blots were stripped and reprobed with anti-β-actin antibody to control for the amounts of protein loaded onto the gel. *P o0.05, **Po 0.01, ***Po 0.001 vs. the vehicle control.

activation of the AhR, Cyp1a1 and Cyp1b1 in mouse neocortical neurons. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and proapoptotic properties of TCS were decreased. To date, only one paper has shown that TCS can interact with the AhR in the rat hepatoma cell line (H4L1.1c4) (Ahn et al., 2008). In that study, it was shown that TCS could activate the AhR to 40% of the level produced by TCDD and could inhibit TCDD activation by 30%, which suggests a competitive interaction between TCS and TCDD (Ahn et al., 2008). One of the functions of the AhR is to control the genes responsible for cell metabolism, including, but not limited to, genes for the enzymes Cyp1a1 and Cyp1b1. Therefore, we aimed to determine the expression of Cyp1a1 and Cyp1b1 at the mRNA and protein levels. Our experiments showed that the expression of AhR and

Cyp1a1 mRNA is significantly decreased in cells exposed to 10 mM TCS for 3 or 6 h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3 h of exposure to TCS, but after 6 h, the mRNA expression of Cyp1b1 was significantly decreased. Unfortunately, the available data concerning the mRNA expression of Cyp1a1 and Cyp1b1 under the influence of TCS are inconsistent. The effect of TCS on the mRNA expression of these enzymes differs depending on the examined tissue, the time interval and the concentration of TCS used. Ku et al. (2014) demonstrated that in the liver of yellow catfish (Pelteobagrus fulvidraco) 24 h of TCS treatment increased the expression of Cyp1a1 mRNA for all tested concentrations. However after 72 and 168 h of exposure to TCS, the opposite effect was observed (Ku et al., 2014). Paul et al. (2009) showed that the effect on the expression of Cyp1a1 mRNA in the liver of TCS-treated rats is dosedependent. They demonstrated that low TCS concentrations caused a decrease in the expression of Cyp1a1 mRNA, whereas high concentrations of TCS increased the expression of Cyp1a1 mRNA (Paul et al., 2009). Similar results have been observed in swordtail fish (Xiphophorus helleri) (Liang et al., 2013). To date, there have been no data concerning the effects of TCS on the expression of Cyp1b1 mRNA. The only study refers to the impact of dioxin TCDD on the expression of Cyp1b1 in murine cerebellar granule neuroblasts. It was found that TCDD caused an enhancement of Cyp1a1 and Cyp1b1 expression at both the mRNA and protein levels (Williamson et al., 2005). As mentioned in the introduction, TCS is structurally similar to TCDD and can be transformed to new chlorinated derivatives, such as 2,8-DCDD, 2,3,7-

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TCDD, 1,2,8-TriCDD and 1,2,3,8-TCDD, during its degradation (Buth et al., 2009; Ding et al., 2015). TCDD has been found to increase the protein expression of Cyp1a1 and Cyp1b1 in many types of cells, such as human lung cancer A549 cells, human and rat mammary epithelial cells and murine cerebellar neurons (Döhr et al., 1997; Chen et al., 2004; Larsen et al., 2004; Williamson et al., 2005). Our data showed that the activity of Cyp1a1 was significantly decreased compared with the control cells following 48 h of exposure to 10 mM TCS. Furthermore, the stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS. In our experiments, the addition of αNF reversed the inhibitory effects of TCS on Cyp1a1 activity. It should be noted that in addition to its AhR antagonistic properties, αNF is also a well-documented inhibitor of metabolic reactions that are carried out by the Cyp1a cytochrome family (Bauer et al., 1995), while we did not observe its inhibitory action when it was given alone to the neocortical neurons. Our results showed that the decrease in activity of Cyp1a1 was accompanied by a decrease in its protein expression level. However, we analyzed the Cyp1a1 mRNA expression 3 and 6 h, but not 48 h after TCS treatment. Similarly, Hanioka et al. (1996) have observed reduced activity of Cyp1a1 in TCS-treated rat liver microsomes. Liang and colleagues have shown that low levels (0.002 mg/L E6.9 nM) of TCS decreased Cyp1a1 mRNA expression in hepatocytes of X. helleri. However, the authors did not observe any changes in Cyp1a1 activity as measured by the EROD method (Liang et al., 2013). Likewise in our study, Ku et al. (2014) have reported decreased enzymatic activity of Cyp1a1 in the liver of P. fulvidraco treated for 24 h with TCS. In contrast to our finding, the authors observed TCSinduced stimulation of Cyp1a1 mRNA expression. Only one paper has shown a strong increase in the activity of Cyp1a1 in human hepatoma HepG2 cells exposed to a 22.2 mM concentration of TCS (Rudzok et al., 2009). The above mentioned data from the literature clearly show that the enzymatic activity of Cyp1a1 and its mRNA expression depend on the applied TCS concentration, the time of exposure and the type of tissue. Expression of constitutive and inducible forms of Cyp1a1 has been recognized as tissuespecific (Chung-Davidson et al., 2004; Di Bello et al., 2007). We demonstrated that in βNF-treated cells the relative concentration of the cytochrome was significantly higher than in TCS-treated neurons. βNF did not enhance Cyp1a1 over the control protein level. We postulate that in our experiments the control level of Cyp1a1 was too high to be stimulated by AhR agonist βNF, possibly because of insufficient level of AhR translocator i.e. ARNT. Moreover, we suggest that high concentration of ROS shown in our experiments might have affected expression of AhR-regulated Cyp1a1 both in βNF- and αNF-treated neurons. In addition, altered function of Cyp1a1 is not always reflected by changes in Cyp1a1 expression. Recently, Hodek et al. (2014) have shown that benzo[a] pyrene enhanced activity but reduced expression level of Cyp1a1 in microsomes from male Wistar rats liver (Hodek et al., 2014). The authors did not observe any effect of βNF and αNF used in cotreatment with benzo[a]pyrene on protein expression of Cyp1a1 which is similar to our results. In this study, TCS diminished Cyp1b1 mRNA and enhanced Cyp1b1 protein expression as detected at 6 and 24–48 h, respectively. The increase in CYP1B1 protein level was observed also in TCDD-exposed human mammary fibroblast (Eltom et al., 1998). In addition to TCS-evoked alteration in Cyp1b1 signaling, we demonstrated that TCS inhibited Cyp1a1 activity and decreased Cyp1a1 mRNA and protein expression levels which were accompanied by diminished AhR mRNA expression as detected at 3 and 6 h of exposure. These effects are not reflected by time-dependent alterations in AhR protein level, possibly because of estrogenic properties of TCS. Indeed, the studies on MCF7 and GH3 cells showed that TCS was able to cause estrogenic effects (Gee et al.,

2008; Jung et al., 2012; Huang et al., 2014). There are several studies concerning the mechanism of CYP1A1 down-regulation by estrogens and estrogen-like compounds (Jeong et al., 2001; Jeong and Kim, 2002; Meucci and Arukwe, 2006; Wójtowicz et al., 2011). Previously Jeong and Kim (2002) demonstrated an impairment of the dioxin-response element (DRE) binding to DNA in Hepa-1c1c7 cells treated with o,p’-DDT which is proven to possess estrogenlike activity. Therefore, we hypothesize that TCS-evoked attenuation of Cyp1a1 activity and reduction of its mRNA and protein levels is due to inhibition of transcriptional activity of AhR. The data on the mechanisms of CYP1A1 down-regulation concern the interactive action of estrogen-like compounds and estrogen receptor with AhR (Meucci and Arukwe, 2006). According to these data, activated estrogen receptor (ER) binds to the xenobiotic response element (XRE) and may either potentiate or repress AhRmediated transcription (Ohtake et al., 2008). Another possibility to explain inconsistency between Cyp1a1 signaling (activity and expression) and TCS-evoked alterations in AhR protein level is a negative correlation between AhR expression and ROS production. According to Barouki and Morel (2001) and Chen et al. (2004), high levels of ROS may decrease expression of AhR and AhR-regulated genes. This could explain the observed paradoxical effect of TCS action, which caused the decrease in AhR-dependent activity and protein expression of Cyp1a1 and the increase in protein level of AhR detected at 24 and 48 h of exposure. Therefore, the next step of our study was to determine the effects of TCS on the production of ROS. Our results revealed that 1 and 10 mM TCS increased the production of ROS and that this effect of TCS was reversed by 10 mM NAC, the ROS scavenger. To date, it has been indirectly demonstrated that TCS increases ROS production in aquatic organisms, such as mussels (Dreissena polymorpha) or Daphnia magna (Binelli et al., 2009; Riva et al., 2012; Sengupta et al., 2015). Recently, it has been described that in rat neural stem cells, 50 mM TCS directly increased the ROS formation and depleted the glutathione activity (Park et al., 2016). High concentrations of ROS are able to affect hypoxia-inducible factor 1-alpha (HIF-1α) and inhibit cyp1a1 expression, most likely through decreasing a pool of aryl hydrocarbon receptor nuclear translocator (ARNT; homolog of HIF-1β) that is necessary for proper function of AhR (Nie et al., 2001). Indeed, AhR was found to interact and crosstalk with HIF-1-mediated signaling e.g. AhR inhibited transcriptional activity of HIF-1 in Hep3B cells, and HIF-1a attenuated transcriptional activity of AhR in Hep3B cells (Chan et al., 1999; Jacob et al., 2015). To confirm an involvement of ROS in TCS-induced toxicity, we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and ROS scavenger. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6 h, respectively. These effects suggest that ROS participate in TCS-evoked effects in mouse neurons, though they could also be result from the action of NAC alone. Our data suggest that NAC-induced increase in Cyp1a1 mRNA might be connected with HIF-1α signaling pathway. The crosstalk of HIF-1α with AhR and limited availability of ARNT which is necessary for AhR signaling pathway can affect transcription of AhR-regulated genes, including CYPs. Additionally, taking into account the involvement of the ROS in the mechanism of TCS action, we cannot exclude that TCSevoked alterations in Cyp1a1 mRNA expression can be also partially regulated by Nrf2/Keap1 transcription factor which is known to interact with AhR signaling pathway (Haarmann-Stemmann et al., 2012). 5. Conclusion Our results are the first to demonstrate that TCS induces AhRdependent apoptosis in neocortical neurons in vitro. We postulate

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that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCSinduced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS. Therefore, further investigations of the mechanisms underlying the effects of TCS on the nervous system are needed.

Acknowledgments This study was supported by Polish National Science Center under Grant No. 2014/13/N/NZ4/04809.

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