n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons

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PHAREP 400 1–10 Pharmacological Reports xxx (2015) xxx–xxx

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Original research article

n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons Gdula-Argasin´ska a,*, Jacek Czepiel b, Justyna Toton´-Z˙uran´ska c,d, Paweł Wołkow c,d, Tadeusz Librowski a, Anna Czapkiewicz f, William Perucki g, Michał Woz´niakiewicz e, Aneta Woz´niakiewicz e

Q1 Joanna

a

Department of Radioligands, Faculty of Pharmacy, Jagiellonian University, Medical College, Krako´w, Poland Department of Infectious Diseases, Faculty of Medicine, Jagiellonian University Medical College, Krako´w, Poland c Department of Pharmacology, Faculty of Medicine, Jagiellonian University Medical College, Krako´w, Poland d Center for Medical Genomics–OMICRON, Jagiellonian University Medical College, Krako´w, Poland e Laboratory of Forensic Chemistry, Faculty of Chemistry, Jagiellonian University in Krako´w, Krako´w, Poland f Faculty of Management, AGH University of Science and Technology, Krako´w, Poland g Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA b

A R T I C L E I N F O

Article history: Received 29 March 2015 Received in revised form 11 August 2015 Accepted 2 September 2015 Available online xxx Keywords: n-3 Fatty acids A549 cells Polycyclic aromatic hydrocarbons Gene expression Isoprostanes

A B S T R A C T

Background: Chronic airway inflammation is coordinated by a complex of inflammatory mediators, including eicosanoids. The aim of this study was to evaluate the impact of polycyclic aromatic hydrocarbons (PAHs) on the human lung epithelial carcinoma A549 cells supplemented with docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids. Methods: We analyzed the influence of DHA, EPA and/or benzo(a)pyrene (BaP), chrysene (Chr), fluoranthene (Flu) and benzo(a)anthracene (Baa) treatment on the fatty acids (FAs) profile and the formation of isoprostanes. We studied the cyclooxygenase-2, FP-receptor, peroxisome proliferatoractivated receptors PPARd and PPARg, transcription factor NF-kB p50 and p65 expression by Western blot, phospholipase A2 (cPLA2) activity, as well as aryl hydrocarbon receptor (AHR), cytochrome P450 (CYP1A1), phospholipase A2 (PLA2G4A) and prostaglandin synthase 2 (PTGS2) gene expression by qRTPCR. Results: DHA or EPA supplementation and BaP or Baa treatment resulted in a higher level of PGF3a. COX-2 expression was decreased while PPARd expression and cPLA2 activity was increased after fatty acid supplementation and PAHs treatment. DHA and EPA up-regulated AHR and PLA2G4A genes. Conclusions: Supplementation with n-3 FAs resulted in changes of inflammatory-state related genes in the lung epithelial cells exposed to PAHs. The altered profile of lipid mediators from n-3 FA as well as repression of the COX-2 protein by n-3 PUFAs in A549 cells incubated with PAHs suggests anti-inflammatory and pro-resolving properties of DHA and EPA. It remains to be shown whether these pleiotropic and protective actions of n-3 FAs contribute to fish oil’s therapeutic effect in asthma. ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.

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Abbreviations: AA, arachidonic acid; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon nuclear translocator; BaP, benzo(a)pyrene; Baa, benzo(a)anthracene; Chr, chrysene cyclooxygenase-2; CYP1 A1, cytochrome P450 1 A1; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; Flu, fluoranthene; MUFA, monounsaturated fatty acids; NF-kB, nuclear transcription factor kB; PAHs, polycyclic aromatic hydrocarbons; PGF2a, prostaglandin F2a; PGF3a, prostaglandin F3a; PLA2G4A, phospholipase A2 IV (cPLA2); PPARs, peroxisome proliferator-activated receptors; PTGS2, prostaglandin synthase 2; PUFAs, polyunsaturated fatty acids; Pyr, pyrene; SFA, saturated fatty acids; UNSFAs, unsaturated fatty acids. * Corresponding author. E-mail address: [email protected] (J. Gdula-Argasin´ska). http://dx.doi.org/10.1016/j.pharep.2015.09.001 1734-1140/ß 2015 Published by Elsevier Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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Introduction

19 Q2 Polycyclic aromatic hydrocarbons (PAHs) are a large family 20 of toxic compounds generated from the combustion of organic 21 materials, diesel exhaust and industrial waste. PAHs exposure has 22 been associated with human cancers in various organs, including 23 skin, lung and bladder. Certain PAHs are metabolized into reactive 24 intermediates and cause DNA damage, as well as lead to the 25 formation of reactive oxygen species [1–4]. 26 Epidemiological studies support the thesis of a positive impact 27 of n-3 polyunsaturated fatty acids (PUFAs) on atopic and asthmatic 28 airway disease. Asthma is a major public health problem, with 29 bronchial inflammation as the therapeutic target. The role of 30 dietary fish oil derived PUFAs in allergic inflammation is 31 controversial. Some findings suggest that dietary fish oil may be 32 beneficial, but it is an ongoing debate, whether long-term 33 supplementation of n-3 PUFA might exhibit more anti-inflamma34 tory capacity [5–8]. Mickleborough et al. demonstrated a protec35 tive effect of fish oil supplementation on exercise-induced 36 bronchoconstriction [8]. It has been hypothesized that variations 37 in asthma prevalence across populations and an increase in the 38 asthma burden seen in westernized societies over the past decades 39 might be related to a combination of a progressively higher intake 40 of n-6 fatty acids (FAs), and a lower intake of n-3 [9]. 41 The beneficial effects of n-3 PUFAs were proven in several 42 observational and experimental studies. PUFAs and their eicosanoid 43 derivatives may play a significant role in modulating the inflamma44 tory response. Lipid mediator metabolomics of self-resolving 45 inflammatory exudates recently highlighted a new family of potent 46 anti-inflammatory and pro-resolving mediators [10,11]. 47 The aim of this study was to evaluate the impact of 48 docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids on the 49 human lung epithelial carcinoma cells (A549) exposed to polycy50 clic aromatic hydrocarbons. 51

Materials and methods

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Cell cultures

53 Human lung carcinoma epithelial cells (A549, ATCC CCL-185) 54 were cultured in F 12K medium with 10% fetal bovine serum, 55 penicillin (100 IU/mL) and streptomycin (100 mg/mL) (ATCC, VA, 56 USA). Cells were maintained at 37 8C in a humidified atmosphere 57 which contained 5% CO2 and were seeded into a 6-well plates 58 Q3 (Sarstedt, Germany), at a density of 5  105 cells/well in 2 mL of 59 medium. During every step of the procedure, cell morphology was 60 investigated by an inverted light microscope (Olympus, Japan). Cell 61 viability during culturing was assessed using the trypan blue 62 exclusion test. 63 A549 cells (24 h after seeding) were incubated with 80 mmol/mL 64 of DHA or EPA (dissolved in ethanol) for 24 h. 10 mmol of BaP, Chr, 65 Flu or Baa (dissolved in dimethylsulfoxide) (Sigma–Aldrich, USA) 66 was added and cells were incubated for 2 h. Control cultures 67 received the same concentration of ethanol (the final content did 68 not exceed 0.12%, v/v) and DMSO (the final content did not exceed 69 0.05%, v/v) as experimental cells.

inhibitor cocktail set III (Calbiochem, Merck, Germany) as described below [12,14]. We used primary antibodies: COX-2, GAPDH (GeneTex, CA, USA), FP-receptor diluted 1:1000 as well as NF-kB p50, NF-kB p65, PPARd and PPARY (Cayman Chemicals, MI, USA) diluted 1:100 in Signal+ for Western blot (GeneTex, CA, USA). The secondary antibody was easy blot anti rabbit IgG (HRP) diluted 1:2000 in Signal+ for Western blot (GeneTex, CA, USA). Proteins were detected using a western blotting detection kit Clarity Western ECL Luminol Substrate (Bio-Rad, CA, USA). The integrated optical density of the bands was quantified using Chemi Doc Camera with Image Lab software (Bio-Rad, CA, USA).

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Quantitative real-time PCR

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RNA was extracted from cells using Maxwell 16 cell LEV total RNA purification kit on Maxwell instrument (Promega, MI, USA). After Q4 quantity and quality evaluation, RNA concentration was normalized to 15 ng/mL. Reverse transcription was done with a high-capacity reverse transcription kit (Life Technologies, NY, USA). qPCR 384wells reaction plate was prepared on Bravo Biorobot (Agilent, CA, USA), performed with TaqMan (Life Technologies) primers and probes for phospholipase A2 IV (PLA2G4A; hCG41183), prostaglandin synthase 2 (PTGS2; hCG39885), aryl hydrocarbon receptor (AHR; hCG17373) and cytochrome P450 (CYP1A1 (hCG2036587) according to the manufacturer’s protocol on CFX384 Touch Real Time PCR Detection System (Bio-Rad, CA, USA). Endogenous control genes (GAPDH, HPRT1, PPIA and TBP) were selected on the basis of the pilot experiment. Relative expression was calculated using DDCq method [13].

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Phospholipase A2 assay

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Cytosolic phospholipase A2 activity was measured in the A549 cells as per manufacturer instructions (Cyaman Chemical). Absorbance after 60 min of incubation was measured at 414 nm. Absorbance changes during the time were calculated using cPLA2Triple software (Cayman Chemical, MI, USA). Data were expressed as mmol/min/mg.

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FA analysis

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Lipid extraction from the cell membranes was carried out using a solution of chloroform/methanol (2:1). The FA methyl esters were analyzed using gas chromatography (Agilent 6890N) with a DB-23 (60 m, 0.25 mm) column as described earlier [14,15]. Results were expressed in relative percentage of the sum of saturated (SFA), monounsaturated (MUFA), polyunsaturated UNSFA, and n-3 and n-6 fatty acids.

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UHPLC/MS-TOF method

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ApoTox-Glo triplex assay

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The ApoTox-Glo triplex assay (Promega, MI, USA) was performed to assess cytotoxicity of the treatment compounds, cell viability and apoptosis, as described previously [12].

Isolation of isoprostanes was done according to the method proposed by Milne et al. [16]. Identification and determination of 8-isoPGF2a, 8-isoPGF3a as well as PGF2a and PGF3a was performed using an UltiMate 3000 RS liquid chromatography system (Dionex, USA) coupled to a mass spectrometer with a time of flight mass analyzer (MicroTOF-Q II, Bruker, Germany). Separation of the studied eicosanoids was carried out on a Synergi 4 m Hydro-RP 80A column (150 mm  2.0 mm I.D., Phenomenex, USA) at 40 8C. PGF3a, 8-iPGF3a, 8-iPGF2a, 5-iPF2a, PGF2a-d9 and 8iPGF2a-d4 standards were obtained from Cayman Chemicals. The details and validation parameters of the method were described previously [14,17].

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Western blot

Statistic

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Cells lysate were prepared using M-PER mammalian protein extraction reagent (Thermo Scientific, IL, USA) with protease

For each analyzed effects from PAHs and FAs treatment the data were obtained as triplicate measurements average from six

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Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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independent experiments. Values are presented as means  SEM. One-way ANOVA with Scheffe’s test was performed to check no differences between kinds of PAHs and for isoP content. Two-way ANOVA was conducted to evaluate the effects from PAHs and FAs treatment. Fatty acids content, proteins or genes expression, respectively were assumed as dependent variables and PAHs treatment and FAs supplementation were exploratory variables. It tested three null hypothesis: H1 – that the means of the dependent variable were equal for different values of PAHs, H2 – the means of the dependent variable were equal for different values of FAs, and H3 – that is no interaction. Calculations were done using STATISTICA 10 (StatSoft Inc., USA) and statistical significance was established as p  0.05.

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Results

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Cells viability

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No cytotoxic effects or apoptosis were observed in the A549 cells treated with DHA or EPA for 24 h and after 2 h of incubation with PAHs. Cell viability varied from 99% to 96% after incubation with PAHs (Fig. 1).

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Western blot

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No differences between kinds of PAHs treatment on the proteins expression in the lung epithelial cells were observed. For COX-2 expression in A549 cells statistical differences were observed for PAHs treatment (F = 72.9, p = 0.000), for FAs supplementation (F = 220.3, p = 0.000) and PAHs  FAs interaction (F = 93.1, p = 0.000) (Fig. 2A). For FP-receptor statistical differences were noted only after FAs supplementation (F = 15.23, p = 0.000) (Fig. 2B). Cytosolic PPARd expression differed statistically in cells after PAHs treatment (F = 161.8, p = 0.000), after FAs supplementation (F = 11.2, p = 0.001) and for PAHs  FAs interaction (F = 11.8, p = 0.001) (Fig. 2C). For the cytosolic PPARg no differences were noted (Fig. 2D). The expression of cytosolic NFkB p50 differed significantly in A549 cells after FAs supplementation (F = 5.76, p = 0.01) and for PAHs  FAs interaction (F = 10.27, p = 0.000) (Fig. 2E). For the cytosolic NF-kB p65, statistical differences were observed in lung epithelial cells after PAHs treatment (F = 60.42, p = 0.000), after FAs supplementation (F = 16.21, p = 0.000) and for PAHs  FAs interaction (F = 7.45, p = 0.008) (Fig. 2F).

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PLA2G4A, PTGS2, AHR and CYP1A1 genes expression

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No differences between kinds of PAHs treatment on the genes expression in the lung epithelial cells were observed. Induction of the AHR gene was statistically different in A549 cells after PAHs treatment (F = 126.0, p = 0.000), after FAs supplementation (F = 300.2, p = 0.000) and for PAHs  FAs interaction (F = 166.0, p = 0.000) (Figs. 3A and 4A). For CYP1A1 expression statistical differences were observed in the lung epithelial cells after PAHs treatment (F = 7.9, p = 0.006), after FAs supplementation (F = 36.34, p = 0.000) and for PAHs  FAs interaction (F = 5.73, p = 0.019) (Figs. 3B and 4B). In the A549 cells supplemented with FAs and activated with PAHs, statistically significant repression of the PLA2G4A gene was noted. After PAHs treatment (F = 73.0, p = 0.000), after FAs supplementation (F = 1036.0, p = 0.000) and for PAHs  FAs interaction F = 26.0, p = 0.000) (Figs. 3C and 4C). For the PTGS2 gene no differences were noted (Fig. 3D).

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cPLA2 activity

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In the lung epithelial cells no differences between kinds of PAHs treatment on the cPLA2 activity were observed. Statistically higher activity of cPLA2 was observed in A549 cells supplemented with FAs (F = 173.6, p = 0.000), after PAHs treatment (F = 3.7, p = 0.058) and for PAHs  FAs interaction (F = 12.3, p = 0.001) (Fig. 5).

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Identification of isoprostanes

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In the control samples, DHA and EPA as well as Chr and Flu supplemented cells absence of the isoprostanes was observed. In A549 cells treated with BaP we observed the presence of 8-isoPGF2a, 8-isoPGF3a and PGF2a. DHA or EPA supplementation and BaP treatment resulted lower levels of 8-isoPGF3a and PGF3a. In the EPA + BaP samples the highest amount of 8-isoPGF3a was observed (12.1 ng/mL). PGF3a content was almost at the same level in the DHA + Bap and EPA + BaP samples, 1.3 ng/mL and 1.1 ng/mL respectively (Fig. 6). We did not detect other lipid mediators, such leukotrienes or pro-resolving mediators in the A549 cells.

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FAs content

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No differences between kinds of PAHs treatment on the fatty acids profile in the lung epithelial cells were observed. SFA content

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Fig. 1. Viability of the A549 cells treated with DHA or EPA (80 mmol) for 24 h before stimulation with PAHs (10 mmol, 2 h). The graphs represent means  SEM from six experiments.

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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differed in A549 cells after FAs supplementation (F = 7.92, p = 0.006) and for PAHs  FAs interaction (F = 7.354, p = 0.008). For UNSFA statistical differences were observed in the lung epithelial cells after FAs supplementation (F = 8.034, p = 0.006) and for PAHs  FAs interaction (F = 7.107, p = 0.009). MUFA content

differed statistically in cells after PAHs treatment (F = 8.72, p = 0.004) and after incubation with FAs (F = 35.8, p = 0.000). n-3 fatty acids profile differed statistically in A549 cells after PAHs activation (F = 43.4, p = 0.000), after incubation with FAs (F = 281.1, p = 0.000) and for PAHs  FAs interaction (F = 14.6, p = 0.000). For

Fig. 2. Relative expression of COX-2 (A), FP-receptor (B), PPARd (C), PPARg (D), NF-kB p50 (E) and NF-kB p65 (F) in the A549 cells supplemented with DHA or EPA before activation with PAHs. The graphs represent six independent experiments.

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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Fig. 2. (Continued )

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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Fig. 2. (Continued ).

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n-6 fatty acids statistical differences were observed after PAHs treatment (F = 91.0, p = 0.000, after FAs supplementation (F = 97.56, p = 0.000). n-3/n-6 ratio differed significantly in the cells after PAHs treatment (F = 91.0, p = 0.000), after FAs supplementation (F = 425.7, p = 0.000) and for PAHs  FAs interaction (F = 46.8, p = 0.000) (Table 1).

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Discussion

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Exposure to PAH results in a variety of molecular responses such as enzyme activation, oxidation, and/or signal transduction [1,3,18,19]. These responses are mainly mediated by the AHR/aryl hydrocarbon nuclear translocator (ARNT) signaling pathway [20,21]. As a ligand for the aryl hydrocarbon receptor, BaP upregulates the expression of phase I bioactivation genes and phase II conjugation genes. Induction of biotransformation enzymes including CYP1A1, CYP1B1 and epoxide hydrolase metabolically activate BaP to different types of metabolites including hydroxylated intermediates, epoxides, quinones, dihydrodiols, dihydrodiol epoxides and various metabolite conjugates in cells [6,22]. In our study incubation with PAHs and FA resulted higher expression of AHR, when compared to control. AHR mRNA expression in the samples supplemented with DHA or EPA and activated with PAHs was up-regulated with statistical significance. In BaP samples, expression of AHR was lower than in the DHA + BaP cells. These observations suggest that both DHA and BaP modulate AHR expression and act as co-regulators. In addition, these observations may suggest cross-talk of AHR with other transcription factors. The interaction of AHR–NF-kB (nuclear transcription factor kB) has become a mechanism linking certain pathological responses induced by environmental insults. Furthermore, the AHR–NF-kB interaction provides a basis for the therapeutic applications of certain AHR ligands to treat human diseases [23]. Investigators have demonstrated that the AHR signaling pathway plays an important role in modulating the immune response in various respiratory diseases and have shown that the respiratory system is sensitive to alterations in AHR expression or function, which also suggest the potential therapeutic effect of AHR ligands [20,23]. In our study there were differences of cytosolic NFkB proteins expression. Significant changes were observed mainly for p65 after PAHs treatment, after FA supplementation and for

PAHs  FA interaction. It suggested that FA inhibited p65 translocation to the nucleus. The toxicity of aromatic hydrocarbons often involves cellular alternations associated with oxidative stress [3,19]. Although many PAHs have been shown to interfere with immune responses, the mechanisms underlying immunotoxicity of these compounds are not fully understood [3,16,24,25]. In our study, cells incubated with DHA or EPA and with PAHs had a decrease of COX-2 expression at the protein level, but no differences of the PTGS2 gene in these samples was also observed. Supplementation with DHA or EPA and subsequent PAHs treatment resulted in the induction of the PLA2G4A gene. The activity of cPLA2 in those samples was significantly the highest, which may demonstrate the synthesis of anti-inflammatory, pro-resolving eicosanoids from DHA and EPA. EPA and DHA compete with AA for release from membrane phospholipids and for metabolism to eicosanoids by COX and LOX. EPA and DHA act as ligands for peroxisome proliferator transcription factor PPARY which inhibits NF-kB signaling. The transcription factor NF-kB promotes immunity by controlling the expression of genes involved in inflammation [10,11]. In our study we observed statistical differences in the cytosolic PPARd protein expression after FA supplementation and for PAHs  FA interaction. Expression of PPARg protein expression remain unchanged. We detected PGF3a in cells supplemented with DHA or EPA and exposed to BaP or Baa. PGF3a is a COX product of EPA. The biosynthesis of PGF3a from EPA was demonstrated in vitro in humans. Little is known about this class of prostaglandin, but we suppose that they may inhibit COX-2 activity. The control of PLA2 activity has been proposed for treating respiratory, inflammatory and allergic diseases. Allergen-induced airway responses show that cPLA2 is an important effector of airway hypereactivity in mice, which may be due to alterations in the downstream products of phospholipid metabolism [24]. Kotha et al. [25] demonstrated that poultry particulate matter induced IL8 secretion by human lung epithelial cells, regulated by cPLA2 activation through extracellular signaling-regulated kinase-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers. Group VI PLA2 enzymes may participate in the repair of oxidized mitochondrial membrane phospholipids [27]. An important feature of cPLA2 is its link to cell surface receptors that stimulate signaling pathways

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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Fig. 3. Relative normalized expression of AHR (A), CYP1A1 (B), PLA2G4A (C) and PTGS2 (D) genes. A549 cells were treated with FAs before activation with PAHs. The graphs represent six experiments.

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associated with activation of protein kinases and production of reactive oxygen species [28]. In A549 cells treated with BaP we observed the presence of 8-isoPGF2a, 8-isoPGF3a and PGF2a. In Baa samples, 8-isoPGF2a

and 5-isoPGF2a were noted. DHA or EPA supplementation and BaP treatment resulted in increased 8-isoPGF3a and PGF3a concentrations. Prostaglandins are produced as a result of COX enzyme activity, isoprostanes are generally thought to form non-enzymatically by

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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J. Gdula-Argasin´ska et al. / Pharmacological Reports xxx (2015) xxx–xxx Table 1 Fatty acid profiles of the A549 cell membranes. Cells were incubated with DHA or EPA and stimulated with PAHs, as indicated. Data expressed as [%]. Means  SEM, n-6. SFA

UNSFA

MUFA

n-6

n-3/n-6

Control Bap Chr Flu Baa

33.7  1.0 30.0  0.9 32.5  0.5 24.4  0.4 32.5  1.2

66.3  1.2 70.0  1.2 67.5  0.9 75.6  0.5 67.5  1.2

35.3  0.9 36.5  0.8 33.1  0.6 41.3  1.0 39.1  0.8

n-3 5.4  0.5 1.7  0.3 3.8  0.4 2.1  0.2 3.2  0.2

25.6  1.1 31.8  0.5 30.6  0.7 32.2  1.0 25.2  0.4

0.2  0.0 0.1  0.0 0.2  0.0 0.1  0.0 0.1  0.0

DHA DHA + BaP DHA + Chr DHA + Flu DHA + Baa

32.0  1.1 32.0  0.7 30.0  1.2 24.9  1.4 23.6  1.1

68.0  1.4 68.0  1.2 70.0  1.3 75.1  1.0 76.4  1.5

31.6  1.2 36.2  1.0 34.0  1.0 30.3  2.0 34.5  0.5

20.9  0.9 8.5  0.6 17.5  0.4 16.7  0.7 19.9  0.9

15.6  0.5 23.3  0.3 18.4  0.5 28.2  1.1 21.9  1.0

1.3  0.2 0.4  0.0 1.0  0.2 0.6  0.1 0.9  0.1

EPA EPA + BaP EPA + Chr EPA + Flu EPA + Baa

24.7  1.0 32.0  1.5 32.0  1.2 30.0  1.0 34.4  0.9

75.3  0.5 68.0  1.1 68.0  2.1 70.0  2.1 65.6  1.5

23.2  0.6 34.1  0.8 29.3  0.8 34.8  0.8 22.2  0.9

31.1  0.7 11.0  0.6 18.2  0.9 13.0  0.5 19.3  0.8

21.0  1.0 23.0  0.5 20.6  0.5 22.2  0.6 24.2  1.1

1.5  0.2 0.5  0.0 0.9  0.2 0.6  0.0 0.8  0.0

SFA – sum of saturated FAs, UNSFA – sum of unsaturated FAs, MUFA – sum of monounsaturated FAs, n3 – sum of n-3 FAs, n6 – sum of n-6 FAs, n-3/n-6 FAs ratio.

Fig. 4. PAHs  FA interactions of the AHR (A), CYP1A1 (B) and PLA2G4A (C) genes expression.

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free radical-mediated peroxidation of AA and other UNSFA. Separate evidence has suggested that COX activity may also contribute to isoprostane production in selected tissues [29–32]. In our previous study, the A549 cells supplemented with EPA or DHA for 24 h and activated with lipopolysaccharide, maresin and protectin D1 were detected [14]. In another study we observed formation of PGF3a, 8-iPGF3a as well as AA derivatives in the HepG2 cells supplemented with EPA and treated with BaP. Our findings suggest that EPA plays a role in the enhancement of anti-oxidant defence [17]. We observed a decrease of FP-receptor expression in the cells after supplementation with FA and after PAHs treatment. Activation of the FP-receptor via 8-iso PGF3a as well as PGF3a which were detected in those samples is suggested. High

expression of the FP-receptor was observed in lung tissue and lung fibroblasts, which facilitates bleomycin-induced pulmonary fibrosis independently of transforming growth factor b [33]. In our study we observed down-regulation of CYP1A1 gene by BaP, Chr and Baa in the lung epithelial cells, while in the cells supplemented with DHA or EPA and treated with Bap and Baa higher CYP1A1 mRNA levels were noticed. Synthesis of CYP eicosanoids is initiated by extracellular signal-induced activation of cPLA2 enzymes that release AA, EPA, and DHA from the sn-2 position of membrane phospholipids. In contrast to the short halflives of prostanoids and leukotrienes, CYP-eicosanoids can be reesterified into phospholipids creating a membrane pool of preformed epoxy and hydroxy-metabolites that is also accessible to PLA2s [33,34]. Higher cPLA2 activity and up-regulation of PLA2G4A gene in the cells supplemented with DHA or EPA and activated with PAHs observed in our study, confirmed this observation. The agonistic/antagonistic properties of PAHs and n-3 FA metabolites thus provide enable further research of chemically and metabolically robust analogs for future in vitro studies. In the presented study there were differences in the FA profile in all groups of cells, those activated by PAHs and those supplemented with FAs. It was found that PAHs-treated cells displayed significantly increased n-6 FA. Our finding suggested the protective role of DHA and EPA on cells exposed to PAHs. Differences in the membrane FA profiles of cells may result from the use of FA for biosynthesis of eicosanoids and from differential expression of genes involved in the synthesis, elongation, and desaturation of FA at the inflammatory state [12,14]. In the study of Barhoumi et al. [22], PUFAs treatment increased BaP membrane accumulation with the greatest increase induced in DHA supplemented cells. It was associated with the induction of phase I (P450) and II metabolizing and detoxifying enzymes. This is in agreement with other studies showing that dietary or media lipid changes quickly alter the PUFAs composition of the membrane phospholipids and that changes in the FA profiles of the membranes might modify the physicochemical environment of the cell sufficiently to affect such functions as receptor activity, enzyme activity or permeability to chemical agents [11,35– 37]. Therefore, alterations in cell membrane FA composition induced by PUFAs appear to be factors underlying differential PAHs metabolism.

Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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Fig. 5. Phospholipase A2 activity [mmol/min/mg] in the A459 cells treated with FAs and activated with PAHs. The graphs represent six experiments.

Fig. 6. Isoprostanes and prostaglandins content in the A549 cells incubated with FAs and activated with PAHs. Data [ng/ml] expressed as means  SEM. Results from six independent experiments. Different letters denote statistical differences, p < 0.001.

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Some studies have shown that diet can modulate the response of organisms to drug absorption and distribution, metabolism and excretion. Diet is also known as a modulator of inflammation and carcinogenesis [37–40]. It has been reported that EPA and DHA are incorporated into cell phospholipids in a time- and dosedependent manner at the expense of AA which is converted into eicosanoids [9–11,38,39]. The current data support the precept that the biochemical basis for the beneficial actions of dietary n-3 PUFAs is the increased synthesis of bioactive oxygenated species rather than the suppression of pro-inflammatory AA-derived products [37,39,40]. n-3 FA, especially EPA and DHA inhibit carcinogenesis. Furthermore, dietary fish oil has recently been shown to play a protective role in PAHs induced carcinogenesis and to significantly reduce levels of DNA – adducts, leading to further reinforcement of the idea that fish oil can be used as an anti-inflammatory as well as cancer chemopreventative agent [5,22,41]. In conclusion, the altered profile of lipid mediators from n-3 fatty acids generated during PAHs exposure in the A549 cells, downregulation of COX-2 protein expression and up-regulation of AHR, CYP1A1 and PLA2G4A genes suggests anti-oxidant, anti-inflammatory and pro-resolving properties of DHA and EPA. DHA and EPA as well as their derivatives are potent pleiotropic anti-inflammatory

signaling mediators that act via mechanisms including the activation of AHR and CYP1-dependent phase 2 gene expression and suppression of pro-inflammatory PTGS2 pathway gene expression. The results of this study have revealed the resolving effect of DHA and EPA. Moreover, these results will guide future efforts to identify ways to reduce the harmful effects of PAHs exposure and ultimately to reduce risk of human living in polluted areas. It remains to be shown whether these pleiotropic and protective actions of DHA and EPA contribute to fish oil’s therapeutic effect in asthma.

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Funding

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This project was possible through the support given by National Q5400 Science Centre, Poland to the author Joanna Gdula-Argasinska 401 DEC-2011/01/B/NZ7/00038. Partially supported by JU MC K/ZDS/ 402 004681. 403 Authors’ contributions

404

Conceived and designed the experiments: JGA; performed the experiments: JGA, AW, JTZ˙; analyzed the data: JGA; contributed

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Please cite this article in press as: Gdula-Argasin´ska J, et al. n-3 Fatty acids regulate the inflammatory-state related genes in the lung epithelial cells exposed to polycyclic aromatic hydrocarbons. Pharmacol Rep (2015), http://dx.doi.org/10.1016/j.pharep.2015.09.001

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reagents/materials/analysis tools: JGA, AW, MW, JTZ˙, PW, TL; statistics: AC, JGA; wrote the paper: JGA, JC, AW, MW, WP.

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Conflict of interest

410

The authors otherwise disclose no conflicts of interest.

411 Q6 Uncited references 412

[26,42].

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