Aryl Hydrocarbon Receptor Activation Contributes to Benzanthrone

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AhR Activation Contributes to Benzanthrone-induced Hyperpigmentation via Modulation of Melanogenic Signalling Pathways Sabiya Abbas, Shamshad Alam, Krishna Pal Singh, Mahadeo Kumar, Shailendra Kumar Gupta, and Kausar Mahmood Ansari Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00364 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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Chemical Research in Toxicology

AhR Activation Contributes to Benzanthrone-induced Hyperpigmentation via Modulation of Melanogenic Signalling Pathways

Sabiya Abbas1,5, Shamshad Alam1, Krishna P. Singh2,Mahadeo Kumar3, Shailendra K. Gupta2,4, Kausar M. Ansari1* 1

Environmental Carcinogenesis Laboratory, Food, Drug, and Chemical Toxicology Group

CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India. 2

Bioinformatics Centre, Systems Toxicology and Health Risk Assessment Group, CSIR-

Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan,31, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India. 3

Animal House Facility, CSIR-Indian Institute of Toxicology Research (CSIR-IITR),

Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India. 4

Department of Systems Biology and Bioinformatics, University of Rostock, 18051 Rostock,

Germany. 5

Department of Biochemistry, School of Dental Sciences, Babu Banarsi Das University

(BBDU) Faizabad Road, Lucknow 226028, Uttar Pradesh, India

Correspondence should be addressed to: Kausar Mahmood Ansari* Email: [email protected]; Tel: +91-522-2627586 ext. 353; Fax: +91-522-2628227

Key Words: Benzanthrone, hyperpigmentation, C57BL/6 mice, AhR receptor, melanogenic signalling 1 ACS Paragon Plus Environment


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Table of Contents (TOC) Graph

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ABSTRACT Benzanthrone (BA), an oxidized polycyclic aromatic hydrocarbon (PAH), has been found to be a potential health threat to occupational workers involved in dye manufacturing factories. It has been observed that occupational workers becomeexposed to BA either during manufacturing, pulverization or storage, and developed various kinds of skin diseases like contact dermatitis, itching, erythema, roughness and

foremost,

hyperpigmentation.

Recently,

it

has

been

shown

that

some

environmental organic pollutants (POPs) like dioxins, furans and polychlorinated biphenyls (PCBs) may act asligands for the aryl hydrocarbon receptor (AhR) and regulate hyperpigmentation. Here, we hypothesized that BA may also act as a ligand for AhR and possibly regulate the melanogenic pathway to induced hyperpigmentation. Our computation results indicate that BA has a high binding affinity towards AhR for the initiation of melanogenic signalling. Following the in silico predictions, we used primary mouse melanocytes (PMMs) and confirmed that exposure to BA (5, 10, and 25 µM) resulted in an increase in AhR expression, tyrosinase activity and melanin synthesis. Moreover, to study the physiological relevance of these findings, C57BL/6 mice were topically exposed to BA and enhanced pigmentation and melanin synthesis were observed. Furthermore, the study was extended to assess the mechanistic aspects involved in BA-induced hyperpigmentation in PMMs as well as in mouse skin. Our results suggest that BA-exposure initiates AhR signalling, increases tyrosinase enzyme activity and melanin synthesis. Moreover, the genes regulating the melanin synthesis, such as TRP-1, TRP-2 and the transcription factor MITF, were also found to be increased. Thus, altogether, we suggest that BA-AhR interactions are critical for BA-induced hyperpigmentation.



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1. INTRODUCTION The skin covers the whole body, forming an effective interface between the body and its environment. Thus, skin is constantly exposed to various toxic environmental factors such as solar ultraviolet radiation (UVR), polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), nitrogen oxides, particulate matter and cigarette smoke.1,2 Therefore, it has been understood that most of the prevalent skin diseases like chloracne, contact dermatitis, and hyperpigmentation have been closely associated with the exposure to environmental contaminants such as polychlorinated biphenyls (PCB), furans, dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and PAHs.3 In this connection, epidemiological studies have shown that benzanthrone (BA), which is an oxy-PAH, is a potential health threat to occupational workers involved in dye manufacturing industries.4 Studies have shown that when these workers were exposed to BA, either during manufacturing, pulverization or storage, they developed various kinds of skin diseases like contact dermatitis, itching, erythema, roughness and foremost hyperpigmentation.5-8 Moreover, authors have demonstrated that exposure to BA followed by chronic exposure to sunlight caused a marked hyperpigmentation in mouse skin.7,8 Although the biochemical basis of BA-induced skin lesions have been studied, knowledge regarding the mechanism of BA-induced skin manifestations are still not clear. Aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor present in many cell types. Following ligand binding, AhR translocates to the nucleus, dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT), enabling the complex to bind to DNA recognition sequences (called dioxin-responsive elements (DREs), and eventually initiate gene transcription.9 Recently, authors have revealed that skin cells expresses the AhR whichacts as a sensor of environmental chemicals. In this regard, they suggested that chemicals such as dioxins, furans and PCB etc. may act as a ligand for AhR and activate the ligand-AhR complex to regulate melanogenesis.10-12 There is also evidence 4 ACS Paragon Plus Environment

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that TCDD, a potent ligand for AhR, and interaction of TCDD-AhR, causes skin hyperpigmentation by inducing tyrosinase activity, a key enzyme responsible for melanin synthesis. The melanin production takes place in melanosomes via activation of three melanogenic enzymes, tyrosinase (TYR), tyrosinase-related protein 1 and 2 (TRP-1 and TRP2) present in the melanosome.10, 13 It was previously established that BA is known to cause hyperpigmentation in animals as well as humans.5,6 However, the molecular mechanisms underlying BA-induced hyperpigmentation has not been established. Considering the similar properties of BA and TCDD, a known inducer of melanogenesis, we hypothesized that one of the mechanisms responsible for hyperpigmentation could be BA-induced AhR signalling. Therefore, in the present study, we first established that BA interacts with AhR using an in silico approach. Moreover, using primary mouse melanocytes (PMMs) and C57BL/6 mice as experimental models, we confirm that BA act as a ligand for AhR and activates downstream signaling cascades regulating tyrosinase activity and melanin synthesis. 2. MATERIALS AND METHODS 2.1. Chemicals and Reagents BA

was

purchased

from

Tokyo

chemical

industry

(Tokyo,

Japan)

ethylenediaminetetraacetic acid disodium salt (EDTA), hydrogen peroxide (H2O2), hoechst, 2-mercaptoethanol, 3, 4-dihroxyphenylalanine (L-DOPA), protease inhibitor cocktail, tris buffer, triton X-100, tween-20, xylene, bovine serum albumin and anti-mouse and antirabbit IgG horseradish peroxidase–linked secondary antibodies were obtained from Sigma Aldrich (St Louis, MO). Primary antibodies to tyrosinase-related protein-1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), tyrosinase Alexa fluor 647 linked secondary antibodies and Fontana-Masson staining kit were procured from Abcam (Cambridge MA, USA). Antibody for β-actin-HRP was purchased from SantaCruz Biotechnology (Santa Cruz, CA). The cDNA synthesis kit was purchased from Applied 5 ACS Paragon Plus Environment


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Biosciences (Foster City, CA,USA) and QuantiTect(R) SYBR Green PCR Kit was procured from Qiagen (Venilo, Netherland). Polyvinylidine fluoride (PVDF) membrane and enhanced chemiluminescence Western Blotting Detection Reagents were purchased from Millipore (Billerica, MA). Super sensitive polymer HRP-IHC detection system kit was purchased from BIO-GENEIX (Fremont, CA USA). All other chemicals and reagents used in this study were of the highest purity commercially available. 2.2. 3D structure of BA PubChem Compound,

one

of

the

linked

databases

within

the NCBI’s

Entrez

information retrieval system was accessed for the retrieval of the 3D structure of BA (PubChem CID:6697). 2.3. Homology modelling of the AhR receptor The 3D modelling of AhR structure was performed using I-TASSER (Iterative Threading Assembly Refinement) server, this server is based on ahierarchical approach for the prediction of protein structure and function. The amino acid residue sequence (sequence GI: 7304873) of the AhR conservative domain (the number of residues was from 278 to 384) for Mus musculuswas obtained from NCBI(http://www.ncbi.nlm.nih.gov/) and modelled by ITASSER using restraints from PDB templates 4WN5, 4F31, 3H82, 1OJ5. I-TASSER server has various different composites modelling to build the full length structure of the protein, mainly by combining the threading, Ab initio modelling and atomic-level structure refinement.14-16 Multiple threading alignments and iterative structure assembly simulation was used to design 3D atomic models.17 Furthermore, assessment of the structural similarity of the model with the template protein structure was done using the template modelling score (TM-score) calculation.18

Eq. 1


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Where L is the length of the target protein, Lali is the number of the equivalent residues in two proteins; di is the distance of the ith pair of the equivalent residues between the two structures. The ‘Max’ means the procedure to identify the optimal superposition matrix that maximizes the sum in Eq. 1. The scale d0 = 3

is defined to normalize the TM-score in a

way that the magnitude of the average TM-score for random protein pairs is independent of the size of the proteins. The implemented TM-score in I-TASSER is a sensitive scale to the global topology for measuring the structural resemblance between two proteins. The TMscore of our AhR top model was 0.86+0.07, which indicates the much better structural match of the target sequence with the templates. Like the TM-score, the confidence score (C-score) determined the accuracy of the predicted structure. This score is defined based on the quality of the threading alignments and the convergence of the I-TASSER’s structural assembly refinement simulations. Eq.2 Where M is the number of structure decoys in the cluster and Mtot is the total number of decoys generated during the I-TASSER simulations, RMSD is the average decoys to the cluster centroid. Z(i) is the Z-score of the best template generated by ith threading in the seven LOMETS programs and Z0(i) is a program-specified Z-score cut off for distinguishing between good and bad templates. The C-score varies between-5 to 2, where the higher the score indicates the better the quality of the generated model. In our case, the C-score of the best-predicted model of AhR using this server was 1.04. 2.4. Molecular docking of AhR protein with BA Before performing docking studies, we prepared both protein and ligand molecules using protocols of Discovery Studio 4.0, respectively. Prepared protein protocol resolved the protein for various problems, such as missing atoms in incomplete residues; missing loop regions; alternate conformations (disorder); nonstandard atom names; incorrect



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protonation state of titratable residues etc. For more valuable docking studies, generation of 3D conformation of BA was attained by the prepare ligands tool of DS 4.0. From experimental literature, we recognized the active site contact residues of the AhR protein which act as an inducer of melanogenesis.19 All these residues were chosen as active site amino acids for our molecular docking studies. Docking

was

performed

using the

CDOCKER protocol of the DS 4.0.20 CDOCKER used soft-core potentials and an optional grid representation to dock ligands into the receptor active site. The generalpurpose of all-atom force field (CHARMm) family of force fields, were applied to protein. Random 10 orientations of BA were generated to improve the docking studies and finally refined by applying low, but most accurate full potential as a refined pose minimization method. 2.5. Isolation and Culture of PMMs PMMs were isolated from newborn C57BL/6 as described previously.21 Briefly, 1 to 2-dayold newborn pups were euthanized/sterilized with betadine (Win Medicare, New Delhi, India) and washed with70% ethanol. The skin was stripped off and floated on 0.25% trypsin–EDTA (Himedia, Mumbai, India) for 2 h at 37°C. The epidermis was separated from the dermis and chopped in melanocyte basal medium containing supplement pack of melanocyte growth medium (PromoCell, Heidelberg, Germany). PMMs at first to third passage were used for all experiments. 2.6. Cell viability of PMMs The effect of BA on the viability of the PMMs was determined by the MTT assay as described by Kumar et al.22 At 70-80% confluence PMMs were treated with different concentrations of BA (1,2.5,5,10, 25 and 50 µM). Twenty four hours after treatment, the MTT assay was performed by adding one tenth solution of MTT dye (5 mg/ml) of the total volume to each well. The 96-wellplate was incubated in a humidified, 5% CO2 incubator at 37ºC for 2 h. Next, 100 µl of the solubilization solution was added to the wells and then the 8 ACS Paragon Plus Environment

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plates placed in the dark for 30 min at room temperature. After brief gentle mixing of contents in each well, the absorbance (OD) of each well was recorded at 550 and 660 nm using a 96-well plate reader (BioTek, USA).The effect of BA on cell viability was assessed as the percentage of cell viability compared with vehicle-treated control cells, which were arbitrarily assigned 100% viability. 2.7. Immunofluorescence staining for AhR in PMMs PMMs (1 x104) were seeded onto a cover slip for immunofluorescence staining of AhR. In brief, cells were exposed to BA (5, 10, and 25 µM) or TCDD (10 nM). After 24 h, PMMs were washed with PBS, fixed with 4% paraformaldehyde. Cells were permeabilized in 0.25% Triton X-100 and subsequently blocked with 1% bovine serum albumin in PBS for 1 h at room temperature. Coverslips were incubated with specific primary antibody against AhR (dilution 1:200) overnight at 4ºC followed by staining with Alexa–Fluor 647 goat anti-rabbit to visualized the primary antibody. Cells were costained with Hoechst for 2 min in the dark, washed and mounted with Vectashield medium (Vector Lab, Burlingame, CA), and viewed under a Leica TCS SPE confocal microscope (Nussloch, Germany) and further processed using ImageJ 1.49v software (NIH, Wayn Rasband, USA). 2.8. Measurement of melanin contents in PMMs The melanin content was determined according to the method of Otreba et al.24 In brief, PMMs (8 × 104 cells/well) were treated with different concentrations of BA (5, 10 and 25 µM) or TCDD (10 nM) for 24 h. Treated PMMs were washed three times with PBS, trypsinised and pelleted by centrifugation. Cell pellets were then dissolved in 100 µl of 1M NaOH at 80°C and then centrifuged for 20 min at 16,000g. The supernatants were placed into a 96-well microplate, and absorbance was measured at 405 nm, a wavelength at which melanin absorbs light. A standard synthetic melanin curve (0 to 400 µg/ml) was performed and melanin content in BA and TCDD treated cells was expressed as the percentage of the controls (untreated PMMs). 9 ACS Paragon Plus Environment


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2.9. Real time -polymerase chain reaction (RT-PCR) PMMs were treated with different concentrations of BA (5, 10, and 25 µM) and TCDD (10 µM) for 24 h. Total RNA was extracted using TRIzol (Ambion, CA, USA) and further onestep RT–PCR kit (Qiagen, Venlo, Netherlands) was used for cDNA synthesis. cDNA was amplified using SYBR green (Qiagen, Venlo, Netherlands)with the following primers: mouse AhR, forward ACC AGA ACT GTG AGG GTT GG, and reverse TCT GAG GTG CCT GAA CTC CT; mouse β actin, forward AGC CAT GTA CGT AGC CAT CC, and reverse CTC TCA GCT GTG GTG GTG AA. Primers were annealed at 60°C and amplified using ABI, QuantStudio 3 and 5 Real-Time PCR Systems (Foster City, CA USA) for a total of 40 cycles. 2.10. Animals and experimental protocol Male C57BL/6 mice (5-6 weeks old) weighing 20 ± 3g were purchased from Charles River Laboratories (Wilmington, MA, USA). After adaptation to the animal facility of CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, they were acclimatized for at least one week, under standard laboratory conditions and given a commercial pellet diet (Provimi Animal Nutrition Pvt. Ltd. Bangalore, India) and water ad libitum. Animals were housed in plastic cages having rice husk as bedding and maintained in a controlled atmosphere at12 h dark/light, 22 ± 2ºC temperature, and 50-60% humidity as per rules laid down by the Animal Welfare Committee of CSIR-Indian Institute of Toxicology Research. All mice were shaved with an electric clipper (Oster, WI, USA) one week prior to the beginning of the experiment and mice showing no signs of hair growth were used for the experiments. All the experiments involving animals were approved by the Institutional Animal Ethics Committee (CSIRIITR/IAEC/23/15), Lucknow. Animals were sacrificed by cervical dislocation with minimal suffering as per CSIR-IITR guidelines. To assess the BA-induced effects in C57BL/6 mice, the animals were divided into three groups having five mice per group. The mice in the first group received a topical 10 ACS Paragon Plus Environment

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application of 0.2 ml acetone, animals in the second group received a topical application of BA (50 mg/kg. b. wt/100µl acetone, daily) and animals in the third group received a topical application of TCDD (125 ng/mouse/100 µl acetone, thrice a week). In our study, we chose the doseof 50 mg/kg b. wt. of BA based on a previous report.25 Similarly, the dose of TCDD (125 ng/mouse/100 µl acetone, thrice a week) used in this study has also been reported earlier.26 The animals of all the groups were sacrificed at 28 days after BA or TCDD application. 2.11. Preparation of cell extracts and western blot analysis Dorsal skin of C57BL/6 mice or treated PMMs werelysed with ice-cold RIPA (RadioImmunoprecipitation Assay) buffer supplemented with protease inhibitors. Forty µg proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA) and incubated with specific primary antibodies at 40C overnight followed by incubation with the HRP-conjugated secondary antibody (Sigma, St. Louis, MO). Bound antibody was detected by enhanced chemiluminescence using ECL Western Blotting Detection Reagents, following the manufacturer’s instructions (Millipore, Billerica, MA). All the blots were stripped and reprobed with β-actin to ensure equal loading of protein. 2.12. Tyrosinase enzyme activity in PMMs and skin lysate Tyrosinase activity in PMMs and skin lysate was determined as per the protocol described by Hunt et al.23 Tyrosinase activity was estimated by measuring the rate of oxidation of LDOPA. PMMs (1 × 105) were treated with BA (5, 10 and 25 µM) and TCDD (10 nM) for 24 h, and subsequently suspended in 50 µl cold 50 mM phosphate buffer, pH 6.8, which contains disodium phosphate and monopotassium phosphateand 1% (w/v) Triton X-100. After pipetting and vortexing to lyse the cells, the extract was clarified by centrifugation at 10,000 rpm for 5 minutes. Similarly, skin tissue was also lysed in cold 50 mM phosphate buffer and clarified by centrifugation at 10,000 rpm for 5 minutes. For assay, fourty microliter of cell or tissue lysate were added to the wells of a 96-well plate and the assay was started by the 11 ACS Paragon Plus Environment


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addition of 100 µl L-DOPA (10 mM) solution at 37°C. Control wells contained 40 µl lysis buffer or boiled cell or tissue lysate. Absorbance at 490 nm was read every minute for at least 20 minutes at 37°C on a microplate reader fitted with a temperature control mechanism (FLUOstar omega, Ortenberg, Germany). Tyrosinase enzymatic activity was expressed as the amount of enzyme transforming 1 µmol of L-DOPA/min/mg of protein. 2.13. Immunohistochemistry of tyrosinase Paraffinembedded 5 µm thick skin sections were deparaffinized and rehydrated with descending concentrations of alcohol. For the detection of tyrosinase expression, rehydrated sections were antigen retrieved in 10 mM sodium citrate buffer (pH 6.0) in a microwave for 5 min at full power. The endogenous peroxidase activity in skin tissue was quenched in 3% H2O2/methanol for 30 minutes at room temperature and rinsed three times in PBS for 5 minute each. The non-specific sites were blocked with 5% goat serum for 10 minutes followed by incubation with anti-tyrosinase rabbit polyclonal antibody (1:200 dilutions) in PBS for 2 h at room temperature in a humidity chamber. The samples were processed with Super Sensitive-IHC detection system (Bio Genex, San Ramon, CA) and incubated with appropriate biotinylated secondary antibody for 30 minutes at room temperature followed by 20 minutes incubation with horseradish peroxidase–conjugated streptavidin. Proteins were visualized with 3, 3’-diaminobenzidine (DAB) for 5-10 minutes at room temperature and counter-stained lightly with Mayer’s haematoxylin for 2 minutes followed by dehydration in ethanol, cleared in xylene and mounted with DPX and scored under a light microscope (Leica Wetzlar, Germany). 2.14. Fontana-Masson’s silver staining Melanin content was examined in skin sections of C57BL/6 mice using a FontanaMassonstain kit (Abcam, Cambridge MA, USA) according to the manufacturer’s protocol with some modifications. In brief, paraffin embedded 5 µm thick skin sections were deparaffinized and rehydrated in descending alcohol concentrations. Next, slides were 12 ACS Paragon Plus Environment

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incubated in warmed ammoniacal silver solution for 60 minutes, rinsed in distilled water and again slides were incubated with 0.2% gold chloride solution 30 seconds. Subsequently, after several washings with distilled water, sections were contrast stained with a nuclear fast red followed by dehydration in ethanol, cleared in xylene and mounted with DPX and observed under a light microscope (Leica Wetzlar, Germany). 2.15. Statistical analysis All the results were expressed as the mean ± S E. Differences between groups were analyzed using one-way ANOVA with Bonferroni intergroup comparison tests from GraphPad Prism3.0 (Graph Pad Prism software, San Diego, CA). A value of, p

Aryl Hydrocarbon Receptor Activation Contributes to Benzanthrone

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