Docosahexaenoic acid attenuates oxidative stress and protects human gingival fibroblasts against cytotoxicity induced by hydrogen peroxide and butyric acid

archives of oral biology 60 (2015) 144–153

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Docosahexaenoic acid attenuates oxidative stress and protects human gingival fibroblasts against cytotoxicity induced by hydrogen peroxide and butyric acid Emilia Zgorzynska a,*, Anita Wierzbicka-Ferszt b, Barbara Dziedzic a, Monika Witusik-Perkowska c, Anna Zwolinska a, Anna Janas b, Anna Walczewska a a

Department of Cell-to-Cell Communication, Medical University of Lodz, Lodz, Poland Department of General Dentistry, Medical University of Lodz, Lodz, Poland c Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Lodz, Poland b

article info

abstract

Article history:

Objective: The oxidative burst of the host cells associated with bacterial pathogen infection

Accepted 25 September 2014

contributes to the destruction of periodontal tissue. The present study investigates the effect of

Keywords:

Methods: The cell viability by MTT assay, ROS level using H2DCF-DA probe, and protein thiol

docosahexaenoic acid (DHA) on human gingival fibroblast (HGF) viability and ROS generation. Docosahexaenoic acid

content were measured in HGFs after 24 h preincubation with different concentrations of DHA

Gingival fibroblasts

followed by treatment with H2O2. The cell death rate was determined by Annexin V/propidium

ROS

iodide staining, andmitochondrial membrane potential (DCm) was examined by MitoTracker Red

Mitochondrial membrane potential

probe in H2O2- and butyric acid-treated HGFs. The fatty acid composition of plasma membranes after incubation with DHA was determined by gas chromatography mass spectrometry. Results: DHA preincubation in a dose-dependent manner increased the viability of HGFs exposed to H2O2 and decreased ROS generation compared to the control cells. In HGFs preincubated with 30 mM DHA, the DCm significantly increased in both H2O2- and butyric acid-treated cells. Moreover, incubation with DHA preserved the protein thiol level as effectively as N-acetylcysteine. Application of 50 mM DHA increased the quantity of viable cells, decreased the number of necrotic cells after H2O2 treatment, and protected HGFs from apoptosis induced by butyric acid. DHA in the plasma membranes of these HGFs represented about 6% of the total amount of fatty acids. Conclusions: These results demonstrate that enrichment of HGFs with DHA reduces ROS generation and enhances the mitochondrial membrane potential protecting the fibroblasts against cytotoxic factors. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Cell-to-Cell Communication, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland. Tel.: +48 422725656; fax: +48 422725652. E-mail address: [email protected] (E. Zgorzynska). Abbreviations: DHA, docosahexaenoic acid; OL, oleic acid; PA, palmitic acid; BA, butyric acid; H2DCF-DA, 20 ,70 -dichlorodihydrofluorescein diacetate; HGF, human gingival fibroblast; ~Cm, mitochondrial membrane potential; MTT, 3[4,5-dimethylothiazol-2-yl]-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species. http://dx.doi.org/10.1016/j.archoralbio.2014.09.009 0003–9969/# 2014 Elsevier Ltd. All rights reserved.

archives of oral biology 60 (2015) 144–153

1.

Introduction

Destructive periodontal disease is multifactorial in its aetiology. Active disease results from a combination of host susceptibility and dental plaque bacteria. Periodontopathogens injure tissue directly, through harmful toxic products that induce cell death and tissue necrosis, and indirectly, through the activation of inflammatory cells which interact with the phagocytic and gum tissue cells.1 During the course of inflammation in response to pro-inflammatory cytokines and mediators, the gingival fibroblasts produce IL-1, IL-6, IL-8, TNFa and TGF-b.2–4 These cytokines recruit polymorphonuclear leukocytes to the site of infection5 which produce proteolytic enzymes and reactive oxygen species (ROS) via the oxidative burst, catalyzed by NADPH oxidase.6 Oxidative DNA damage in fibroblasts and increased hydrogen peroxide production in polymorphonuclear leukocytes have been reported in induced periodontitis in animal studies.7,8 Clinical studies in patients with periodontitis demonstrated elevated levels of oxidative stress markers in saliva, serum and gingival crevicular fluid.9,10 Moreover, positive correlations were observed between periodontal parameters and lipid peroxidation in crevicular fluid and saliva.11,12 Overwhelming evidence indicates that oxidative stress play a central role in the periodontal tissue and alveolar bone destruction.13–15 Docosahexaenoic acid (DHA, C22:6), n-3 unsaturated fatty acid (n-3 PUFA), has potent anti-inflammatory effects. It has been demonstrated that exposure of endothelial cells to DHA reduced expression of COX-2 and NADPH oxidase 4, blocked nuclear p65 NF-kB subunit translocation into the nucleus, and diminished IL-1-stimulated ROS generation.16,17 DHA has also been reported to have a dose-dependent effect on GSH content and antioxidant enzyme activity.18 The action of DHA in the counter-regulation of inflammation seems to be complex. DHA, being highly unsaturated, can be converted by various lipoxygenases to bioactive di- and trihydroxy derivatives, resolvins and protectins, which promote resolution of inflammation.19 When added to the cell culture medium and incorporated into membrane phospholipids,18,20 DHA may compete with arachidonic acid and attenuate chronic inflammatory diseases by reducing pro-inflammatory mediator production.21 Although DHA has only been reported to be present in significant amounts in the retina and neurons,22 dietary supplementation with fish oil caused a rapid incorporation of n-3 PUFA into neutrophils and the epidermis.23 Furthermore, a cross-sectional study of the U.S. adult population suffering from periodontitis demonstrated that increased dietary DHA was associated with a lower prevalence of periodontitis.24 Therefore, an assessment of the role of DHA in the protection of gingival fibroblasts against cytotoxic factors was conducted. Hydrogen peroxide (H2O2) and butyric acid were chosen as the cytotoxic compounds. Physiologically, hydrogen peroxide is produced in abundance by NADPH oxidase in stimulated neutrophils, and can locally reach micromolar concentrations.25 Some toothpastes and mouth rinses contain low concentrations of hydrogen peroxide as a disinfectant to prevent plaque and inflammation of the gums. However, hydrogen peroxide at higher concentrations is a whitening

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ingredient in many home-use and in-office use tooth whitening products.26–28 Hydrogen peroxide is harmful to cellular proteins, and in the presence of transition-metal ions29 is a source of more reactive hydroxyl free radicals (OH), which strengthen the harmful effect of oxidants.30,31 Butyric acid, the local metabolite of pathogenic periodontal bacteria can induce caspase-dependent apoptosis in gingival fibroblasts.32 Here, we report that enrichment of HGFs with DHA attenuates oxidative stress and protects the cells from death induced by both cytotoxic compounds.

2.

Materials and methods

2.1.

Reagents

Dulbecco’s modified Eagle’s medium (DMEM), DMEM without phenol red, fetal bovine serum (FBS), penicillin/streptomycin, phosphate buffered saline (PBS), and trypsin/EDTA were purchased from Biochrom AG (Deutschland); ProLong Gold antifade reagent with DAPI, MitoTracker Red CMXRos, DTNB (5,50 -dithio-bis-(2-nitrobenzoic acid), L-cysteine, and 20 ,70 dichlorodihydrofluorescein diacetate (H2DCF-DA) were obtained from Invitrogen (Poland); M-PER mammalian protein extraction reagent was from Pierce (Rockford, IL); Annexin V Binding Buffer, Annexin V-FITC were purchased from BD Biosciences (Poland); Hydrogen peroxide (30%) came from Chempur (Poland); 3-[4,5-dimethylothiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), formaldehyde, N-acetyl-Lcysteine (NAC), propidium iodide (PI), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), palmitic acid (16: 0), oleic acid (18: 1n-9) and docosahexaenoic acid (22: 6n-3) were obtained from Sigma Aldrich (Poland). Sensitive to oxidation DHA was dissolved in ethanol to make 100 mM stock solution, and stored at 80 8C in a nitrogen atmosphere. The stock solutions were diluted to reach a final concentration immediately before use.

2.2.

Cell culture

Primary human fibroblasts were obtained from gingival tissue of healthy patients undergoing extraction of the third molar after their informed consent. The protocol of the study was approved by Ethics Committee of the Medical University of Lodz. The gingival fragments were cut into small pieces (2 mm  2 mm) and rinsed several times in PBS with antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml and amphotericin 2.5 mg/ml). The tissue was then cultured in DMEM supplemented with 10% FBS, and 1% penicillin– streptomycin solution in Petri dishes. The growth medium was changed every two to three days. When the cells surrounding the tissue explants had formed a confluent monolayer, they were washed with PBS w/o Ca2+ and Mg2+, and detached using 0.25% trypsin – EDTA (0.02% in PBS). The cells were collected by centrifugation at 200  g for 5 min and the pellet was dispersed in DMEM with 10% FBS. Viable cells were counted using trypan blue dye, transferred into 25-cm2 or 75-cm2 cell culture flasks and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 8C. Cells from passages 3–7 were used for all of the experiments.

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2.3.

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Cell viability assay

The colorimetric MTT assay based on the cleavage of yellow tetrazolium salt (MTT) to purple formazan was used to determine HGF viability. Briefly, cells were seeded in 96-well flat-bottomed microplates at a density of 2  104 cells/well in 200 ml of DMEM. After 48 h cells were washed and treated with H2O2 at concentrations ranging between 0.23 and 7.5 mM for 2 h. As controls, cells cultured in FBS-free DMEM for the IC50 of H2O2 determination and in 0.03% EtOH in all other experiments were used. To determine the effect of DHA on the HGF viability cells were preincubated with DHA at concentrations between 5 and 50 mM for 24 h, subsequently exposed to H2O2 at concentrations of 0.23, 0.45, 0.9, 1.8, 3.7, and 7.5 mM for 2 h, then, washed and incubated with MTT (0.5 mg/ml) in darkness. The formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using multilabel plate reader (Victor2, Perkin-Elmer). The experiments were repeated three times in triplicate. Cell viability was calculated as a percentage of viable cells in H2O2-treated groups with respect to viable cells in the control medium.

2.4.

Intracellular reactive species detection

The intracellular ROS were determined with 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCF-DA) probe. Fibroblasts were cultured in 24-well plates at a density of 5  104 cells/well in 700 ml of DMEM for 48 h. Then medium was replaced with 10, 30 and 50 mM DHA, or oleic and palmitic acids (30 mM) in FBS-free DMEM for 24 h. At the end of the incubation, the cells were loaded with 10 mM H2DCF-DA for 30 min. After two washes with PBS, H2O2 in phenol red-free DMEM was added, and fluorescence of 20 ,70 -dichlorofluorescein (DCF)33 was measured in the plate reader (Ex/Em = 488/530 nm) three times in triplicate.

2.5.

Protein thiols determination

Protein thiol level was determined using DTNB (5,50 -dithio-bis(2-nitrobenzoic acid). DTNB reacts with free sulfhydryl groups and gives a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) measurable yellow-coloured product.34 Fibroblasts were seeded at a density 1  106 cells in 6 cm Petri dishes and culture in DMEM for 48 h. The medium was then replaced with 50 mM DHA or 2 mM N-acetylcysteine (NAC) in FBS-free DMEM. After 24 h the cells were treated with 0.9 mM H2O2 for 2 h, then washed with PBS, and lysed with ice-cold M-PER mammalian protein extraction reagent. The total protein content was determined by a Lowry’s method using BSA as a standard.35 The thiols determination was performed according to the manufacturer’s instruction. Briefly, cell lysates were mixed with DTNB in freshly prepared deoxygenated buffer (pH 7.6) containing 40 mM sodium phosphate and 2 mM EDTA. The concentration of sulfhydryl groups was measured at 412 nm in UV-Vis spectrophotometer in triplicate, and calculated in micromoles per milligram of protein using the standard curve of L-cysteine.

2.6.

Annexin V/propidium iodide assay

The quantification of apoptotic and necrotic cells was performed by Annexin V/propidium iodide (PI) double staining

and detected by flow cytometry. Fibroblasts were seeded in 12well plates at a density of 1  105 cells/well in 1.5 ml of DMEM. After 48 h medium was replaced with 50 mM DHA in FBS-free DMEM. Twenty-four hours later, the fibroblasts were treated with 0.9 mM H2O2 or 10 mM butyric acid for 2 h. HGFs were harvested with trypsin–EDTA, rinsed with PBS, re-suspended in 1 binding buffer with 5 ml Annexin V-FITC and 1 ml PI (500 mg/ml), and incubated in darkness at room temperature for 15 min. Then, fluorescence intensity of each population of 10 000 cells was measured in FACSCanto II flow cytometer (Becton Dickinson) and analyzed using BD FACSDiva software. The experiments were repeated three times in duplicate.

2.7.

Mitochondrial membrane potential assessment

The lipophilic mitochondrion-selective fluorescent probe, MitoTracker Red CMXRos, was used to assay the mitochondrial membrane potential (DCm).36 Briefly, HGFs were grown on coverslips in 24-well plates at a density 3  104 cells in 700 ml of DMEM for 48 h. Cells were then incubated with 30 mM DHA in FBS-free DMEM for 24 h followed by exposure to 0.45 mM, 0.9 mM H2O2 or 3 mM butyric acid. After 2 h, HGFs were washed and loaded with 200 nM MitoTracker Red in PBS for 30 min. The cells attached to coverslips were fixed with 4% paraformaldehyde in PBS and mounted using ProLong Gold antifade reagent with DAPI. Fluorescence images from three experiments were then obtained (Ex/Em = 579/599 nm) with Olympus BX-41 microscope. All micrographs were taken under the same conditions without any correction of the captured images.

2.8.

Cellular fatty acid determination

Fatty acid composition in the membranous fraction of HGFs after incubation with DHA was determined by gas chromatography mass spectrometry (GC/MS). Fibroblasts were seeded in 10-cm Petri dishes at a density of 2  106 cells in DMEM, and incubated with 50 mM DHA in FBS-free DMEM. Twenty-four hours later, the cells were washed with PBS w/o Ca2+ and Mg2+ three times, detached with trypsin, and pelleted by centrifugation at 600  g for 5 min at 4 8C. Then, the fibroblasts were re-suspended in an ice-cold buffer (225 mM mannitol, 75 mM sucrose, 0.05 mM EDTA and 30 mM Tris–HCl) and homogenized in pre-cooled glassware with a pestle at 4 8C. The homogenates were then centrifuged (1000  g for 5 min at 4 8C). Pellets containing the nuclei were left, and the supernatants were centrifuged at 10 000  g for 10 min at 4 8C. The pellets containing mitochondria and fragmented membranes were combined with the nuclei pellet. Lipids from the membranous fraction were extracted using a modified Folch procedure.37 Briefly, a sample with chloroform:methanol (2:1, v/v) containing 0.02% butylated hydroxytoluene (BHT) as an antioxidant was mixed with onefifth the volume of 0.58% NaCl, and the organic and aqueous phases were allowed to separate at 4 8C all night. The lipid extracts were dried under nitrogen and subsequently converted into fatty acid methyl esters. The fatty acid methyl esters composition was determined at the National Food and Nutrition Institute (Warsaw, Poland) according to protocol no. 10, version 2, 09/03/2009.

archives of oral biology 60 (2015) 144–153

2.9.

147

Statistical analyses

The results are presented as the means with their standard errors. The statistical comparison of groups incubated with and without DHA was performed by ANOVA with Bonferroni correction. For nonparametric data, analyses were conducted using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. To compare the means of two independent values the two-tailed Student’s t test was used. All of the statistical analyses were conducted using Statistica for Windows version 10.0 (StatSoft, Poland). The level of significance was set at p < 0.05.

3.

Results

3.1.

DHA increases HGF viability after H2O2 treatment

The viability of HGFs was not affected by DHA at any concentration used in the experiments (data not showed). The half inhibitory concentration of H2O2 (IC50) was calculated to be 0.69 mM. The viability of HGFs treated with H2O2 at concentrations: 0.45, 0.9 and 1,8 mM varied between 14% and 84%. Twentyfour hour incubation with DHA at concentrations of 30 mM and 50 mM most effectively increased the viability of cells treated with H2O2 ( p < 0.01 and p < 0.001, respectively) compared to the cell viability without preincubation with DHA (Fig. 1a–c). Moreover, 10 and 20 mM of DHA increased the viability of fibroblasts treated with 1.8 mM H2O2 which reduced the cell viability of the control HGFs about 86% (Fig. 1c)

3.2.

DHA inhibits ROS generation

Preincubation of fibroblasts with DHA significantly reduced intracellular ROS level depend on DHA concentration in culture medium (Fig. 2). DHA at a concentration of 10 mM was ineffective in reducing of ROS level within 24 h after H2O2 treatment. DHA at a concentration of 30 mM significantly reduced ROS level in equal measure in HGFs treated with both H2O2 concentrations after the 5th up to 24th hour (Fig. 2a and b). DHA at a concentration of 50 mM reduced ROS level in the greater extent than 30 mM, and faster, from the third hour of treatment with 0.45 mM H2O2 and right from the first hour when cells were treated with 0.9 mM H2O2 ( p < 0.0001 and p < 0.05, respectively). Incubation of HGFs with oleic acid (OL) and palmitic acid (PA) at the same concentration as DHA confirmed specificity of DHA in inhibition of ROS production (Fig. 2c). Only DHA caused a significantly lower ROS level; from the 3rd up to 24th hour. However, there was a slight decrease in DCF fluorescence at 24th hour in HGFs incubated with PA.

3.3.

DHA protects protein thiol level after H2O2 treatment

The concentration of cysteine was used as an index of intracellular protein oxidation by H2O2. As shown in Fig. 3, cysteine concentration decreased from 11.7 mmol/mg protein

Fig. 1 – The viability of H2O2-treated human gingival fibroblasts (HGFs) preincubated with DHA. HGFs were seeded in 96-well plates at a density of 2 T 104 cells/well for 48 h. Cells were then incubated with 0, 5, 10, 20, 30 and 50 mM DHA for 24 h and exposed for 2 h to H2O2 at concentrations: (a) 0.45 mM; (b) 0.9 mM; and (c) 1.8 mM. Values are means W SEM of three independent experiments, each in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to cells without preincubation with DHA.

in the control cells to 7.3 mmol/mg protein in fibroblasts treated with 0.9 mM H2O2 ( p < 0.05). Twenty-four hour incubation of HGFs with 30 mM DHA prior to H2O2 treatment significantly increased the cysteine content to 11.2 mmol/mg protein ( p < 0.05), and was almost equally effective in the conservation of cysteine as a 24-h preincubation with 2 mM NAC (11.9 mmol/mg protein; p < 0.01).

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Fig. 3 – The protein thiols content in H2O2-treated HGFs preincubated with DHA or N-acetylcysteine (NAC). HGFs were seeded in Petri dishes at a density of 1 T 106 cells for 48 h then incubated with 30 mM DHA or 2 mM NAC for 24 h followed by a 2-h treatment with 0.9 mM H2O2. Control cells were cultured in DMEM. Bars are means of L-cysteine concentration W SEM from three independent experiments, each in triplicate. #p < 0.05 compared to the control cells; *p < 0.05, **p < 0.01 compared to H2O2-treated cells without preincubation with DHA or NAC.

3.4. DHA protects HGFs from cytotoxicity induced by H2O2 and butyric acid Preincubation of HGFs with 50 mM DHA followed by treatment with 0.9 mM H2O2 increased the percentage of viable cells (Annex/PI) by 24.5% and decreased the percentage of late apoptotic and necrotic cells (Annex+/PI+ and Annex/PI+) by 23.3% (Fig. 4f) In contrast to hydrogen peroxide, butyric acid caused a marked induction of apoptosis in HGFs. Preincubation with 50 mM DHA followed by treatment with 10 mM butyric acid resulted in a 13.5% decrease in the number of late apoptotic and necrotic cells (Annex+/PI+) and tendency to increase in the quantity of the viable cells.

3.5. DHA enhances mitochondrial membrane potential in H2O2- and butyric acid-treated HGFs

Fig. 2 – The ROS generation in H2O2-treated HGFs preincubated with or without DHA. Cells were seeded in 24well plates at a density of 5 T 104 cells/well for 48 h and preincubated with 10, 30 and 50 mM DHA for 24 h. Then, cells were loaded with H2DCF-DA and treated with: (a) 0.45 mM; (b) 0.9 mM H2O2; and (c) HGFs were preincubated with DHA, oleic acid (OL) and palmitic acid (PA) at the same concentration (30 mM) or in DMEM without fatty acid (control). Values are represented as mean DCF fluorescence W SEM of three independent experiments, each in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared to H2O2-treated cells without preincubation with DHA at the same time points; ^p < 0.0001 compared to the control cells at the same time point.

As the images shown (Fig. 5a) in HGFs cultured in the control medium and with DHA alone, the filamentous mitochondrial network was clearly visible and filled the entire cell volume. The cytotoxic compounds hydrogen peroxide and butyric acid differentially affected the mitochondrion network and significantly decreased DCm compared to the cells incubated in the control medium (Fig. 5b). In fibroblasts treated with 0.9 mM H2O2 the mitochondrial network was barely visible with small spheres which indicate the impairment of oxidative phosphorylation. Incubation of HGFs with butyric acid caused the filaments to be thinner with an increased dissipation of DCm compared to cells grown in the control medium. However, preincubation with 30 mM DHA significantly increased the MitoTracker fluorescence in both H2O2 and butyric acid treated HGFs (Fig. 5b) but in H2O2-treated fibroblasts preincubated with DHA the fluorescence increased about 20% above the fluorescence in the control cells while in BA-treated cells

archives of oral biology 60 (2015) 144–153

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Fig. 4 – Assessment of the cell death rate induced by H2O2 and butyric acid in HGFs preincubated with or without DHA. Representative FACS scatters of (a) control cells; (b) H2O2-treated cells; (c) butyric acid-treated cells; (d) cells preincubated with DHA and treated with H2O2; and (e) cells preincubated with DHA and treated with butyric acid. HGFs were seeded in 12-well plates at a density of 1 T 105 cells for 48 h, then were incubated with 50 mM DHA for 24 h followed by 2-h treatment with 0.9 mM H2O2 or 10 mM butyric acid. HGFs were stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. The graph (f) shows percentage of viable cells (AnnexS/PIS), early apoptotic cells (Annex+/PIS), late apoptotic/necrotic cells (Annex+/PI+), and necrotic cells (AnnexS/PI+). Values are means W SEM of three independent experiments, each in duplicate. *p < 0.05, ****p < 0.0001 compared to the each population of the control cells; ##p < 0.01, ### p < 0.001 compared to H2O2-treated cells without preincubation with DHA; ^^p < 0.01 compared to butyric acid-treated cells without preincubation with DHA.

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Fig. 5 – Representative images of HGFs stained with MitoTracker Red CMXRos and DAPI, magnification 40T (a). HGFs were seeded at a density of 3 T 104 cells on coverslips and grown in 24-well plates for 48 h, then incubated with or without 30 mM DHA followed by 2-hour treatment with 0.45 mM H2O2, 0.9 mM H2O2 or 3 mM butyric acid (BA). The graph (b) shows the mean fluorescence intensity (MFI, arbitrary units). Bars represent mean fluorescent intensity of MitoTracker Red W SEM. The experiments were performed three times, each in triplicate. Asterisk depicts statistical significance relative to control, and triangle relative to treated cells without DHA preincubation. All differences are at the level of p < 0.0001.

preincubated with DHA the fluorescence of the mitochondrion increased to the level in the control cells.

3.6.

in oleic (C18:1) and saturated C13:0 fatty acids. There was also a slight increase in content of two monounsaturated fatty acids, palmitoleic (C16:1) and heptadecenoic (C17:1).

Fatty acid composition

The gas chromatography revealed an incorporation of DHA into membrane lipids and the rearrangement of fatty acid composition. DHA was not detected in the membranes of cells grown in the control medium but in membranes of HGFs incubated with DHA constituted 6% (v/v) of the total fatty acids (Table 1). The increased DHA content in HGFs was linked with a decline in the following fatty acids: palmitic (C16:0), stearic (C18:0), and dihomo-gamma-linolenic (C20:3), and an increase

4.

Discussion

In the present study, the incubation of HGFs with DHA enriched plasma membranes with bioactive n-3 PUFA and counteracted the cytotoxic effects of H2O2 and butyric acid. As expected, the both of cytotoxic agents elicited HGF death and reduced the DCm. Hydrogen peroxide used at concentrations of 0.0015% (0.45 mM) and 0.003% (0.9 mM) considerably

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Table 1 – Fatty acid composition in gingival fibroblasts incubated with DHA. Fatty acid 13:0 14:0 15:0 16:0 18:0 16:1 17:1 18:1 20:3 n-6 22:6 n-3

Control

DHA

Mean  SEM

Mean  SEM

36.00  0.129 0.75  0.005 0.91  0.019 18.70  0.132 26.40  0.199 0.92  0.018 1.24  0.015 13.40  0.178 1.78  0.017 –

51.00  0.840* 0.62  0.010 0.55  0.015 9.14  0.075* 9.34  0.130* 1.67  0.020# 2.02  0.055# 18.60  0.455* 0.88  0.020$ 6.15  0.090*

Values are percentages of total fatty acids  SEM. One-way ANOVA with Bonferroni correction was used to compare the differences between the control cells and DHA-treated cells. # p < 0.05. $ p < 0.01. * p < 0.0001

increased the number of cells labelled with Annexin V/ propidium iodide and with propidium iodide alone, indicating that the permeability of the cellular membrane had been augmented.38 In contrast, periodontopathogen butyric acid, elicited the HGF apoptosis designated by phosphatydyloserine (PS) translocation, detected by Annexin V.39 However, preincubation of HGFs with DHA markedly reduced the number of cells undergoing death due to both apoptosis and increased membrane permeability elicited by the cytotoxic compounds. DHA is taken up from extracellular fluid and transported across plasma membranes into cells by the fatty acid transport proteins and FAT/CD36 translocase.40,41 It has been demonstrated that increased delivery of DHA into the cells promotes the synthesis of PS42 which facilitates docking of pyruvate dehydrogenase kinase and PKB/Akt by pleckstrin homology domains (PH) and enhances the PI3K/Akt signalling pathway.43 Thus, enrichment of HGF plasma membranes with DHA accompanied by reorganization of the lipid microdomains44 promotes the production of intracellular survival signals, and may rescue HGFs from necrosis induced by H2O2 at concentrations of less than one mole. This concentration is about three orders of magnitude less than commercially available tooth bleaching products which may present a risk to the oral mucosa or even the connective tissue if they remain in contact with the gum for longer. The HGFs preincubated with DHA and treated with H2O2 and butyric acid revealed elevated hyperpolarization of the mitochondria showed by greater sequestration of MitoTracker. Under physiological conditions, the mitochondria are implicated in cellular homeostasis and survival.45 As exocitotoxic stimuli cause profound mitochondrial membrane depolarization, with opening of the permeability transition pore being manifested as apoptosis activation and abnormalities of intracellular calcium metabolism, DCm has been often used as a marker for cell viability.46,47 Cardiolipin, an anionic phospholipid present almost exclusively in the inner mitochondrial membrane,48 represents about 15% of the total mitochondrial phospholipids,49 and directly confers fluidity and stability of the mitochondrial membrane.50 A loss of

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cardiolipin decreases the cytochrome c binding to the inner mitochondrial membrane favouring its release.51 It has been showed that mitochondrial cardiolipin can be enriched with DHA,52 thus, increased content of cardiolipin or spatial modification of the mitochondrial membrane by DHA could be responsible for promoting cell viability and survival likely by sequestering cytochrome c and inhibiting pore formation.53 It has been demonstrated that glutathione peroxidase (GPx) deactivates both extracellular and intracellular H2O2,54,55 and that increased DHA content in membrane phospholipids upregulates GPx expression in non-cancer cells.56 Moreover, Arab and co-workers showed increased GSH content and GSHsynthesizing enzyme activity to be present in fibroblasts incubated with DHA.18 In our study, the intracellular generation of ROS in HGFs enriched with DHA was measured within 24 h from the time point at which increased activity of the GSH-dependent redox system was determined in Arab et al. study. In addition, the progressive inhibition of intracellular ROS level began, at the earliest, one hour after exposure of HGFs to H2O2, which may indicate that the GSH pool alone could not be sufficient to sustain ROS elimination within 24-h cell culture. Furthermore, in the present study, a marked increase in redox potency was revealed by the MTT assay, to a great extent executed by succinate dehydrogenase,57,58 and the preservation of protein thiols in H2O2-treated HGFs enriched with DHA. More likely, DHA incorporated into plasma membranes increased the cellular redox potency, in part by restoring the mitochondrial membrane potential. It is commonly accepted that the efficiency of electron flow along the respiratory chain depends directly on the reduction status of the respiratory chain carriers.59 Succinate dehydrogenase anchored to the inner mitochondrial membrane is responsible for both the delivery of electrons to complex III and the control of the ubiquinone pool providing the reduced equivalents into the respiratory chain.60 This may indicate that an augmentation of the mitochondrial succinate:ubichinon reductase activity in the HGFs enriched with DHA in part, was responsible for enhancing the intracellular reduction potency and, as well as the elevation of the DCm, contributing to ROS scavenging and promotion of HGF survival. In summary, the present results show that the enrichment of HGFs with DHA diminished ROS production for up to 24 h, promoted HGF viability and rescued cells from death induced by H2O2 and butyric acid. These findings may clarify the results of the double-blind, placebo-controlled, randomised trial carried out in India and published in this year which confirmed n-3 PUFA benefit in chronic periodontitis.61 These findings indicate that these bioactive fatty acids could act as potential therapeutic adjuncts for the clinical management of periodontal diseases. However, further investigations are required to elucidate the specific roles of individual n-3 fatty acids, DHA and eicosapentaenoic acid (EPA).

Funding This work was supported by Medical University of Lodz, Poland, contract No. 503/0-079-04/503-01.

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Competing interests The authors declare no conflict of interest.

Ethical approval The protocol of the experiment was reviewed and approved of by the Ethics Committee of the Medical University of Lodz (RNN/121/11/KE).

Authors contribution All authors have contributed to the paper and agree with the present version of the paper. A.W. designed the study. E.Z., A.W., A.W.-F., M.W.-P, A.Z., B.D. contributed to collection of data, laboratory work and data interpretation. A.J. provided the gingival tissue from patients. A.W., E.Z., B.D. prepared the manuscript.

Acknowledgements The authors would like to thank Prof. Czeslaw Cierniewski for cooperation and Leslaw Miskiewicz for preparation of image files.

references

1. Page RC, Kornman KS. The pathogenesis of human periodontitis: an introduction. Periodontol 2000 1997;14:9–11. 2. Imatani T, Kato T, Okuda K. Production of inflammatory cytokines by human gingival fibroblasts stimulated by cellsurface preparations of Porphyromonas gingivalis. Oral Microbiol Immunol 2001;16(2):65–72. 3. Morandini AC, Sipert CR, Ramos-Junior ES, Brozoski DT, Santos CF. Periodontal ligament and gingival fibroblasts participate in the production of TGF-b, interleukin (IL)-8 and IL-10. Braz Oral Res 2011;25(2):157–62. 4. Yucel-Lindberg T, Brunius G. Epidermal growth factor synergistically enhances interleukin-8 production in human gingival fibroblasts stimulated with interleukin-1b. Arch Oral Biol 2006;51(10):892–8. 5. Kobayashi Y. The role of chemokines in neutrophil biology. Front Biosci 2008;13:2400–7. 6. Giannopoulou C, Krause KH, Mu¨ller F. The NADPH oxidase NOX2 plays a role in periodontal pathologies. Semin Immunopathol 2008;30(3):273–8. 7. Tomofuji T, Azuma T, Kusano H, Sanbe T, Ekuni D, Tamaki N, et al. Oxidative damage of periodontal tissue in the rat periodontitis model: effects of a high-cholesterol diet. FEBS Lett 2006;580(15):3601–4. 8. Ekuni D, Tomofuji T, Tamaki N, Sanbe T, Azuma T, Yamanaka R, et al. Mechanical stimulation of gingiva reduces plasma 8-OHdG level in rat periodontitis. Arch Oral Biol 2008;53(4):324–9. 9. Takane M, Sugano N, Ezawa T, Uchiyama T, Ito K. A marker of oxidative stress in saliva: association with periodontallyinvolved teeth of a hopeless prognosis. J Oral Sci 2005;47(1):53–7.

10. Wei D, Zhang XL, Wang YZ, Yang CX, Chen G. Lipid peroxidation levels, total oxidant status and superoxide dismutase in serum, saliva and gingival crevicular fluid in chronic periodontitis patients before and after periodontal therapy. Aust Dent J 2010;55(1):70–8. 11. Tsai CC, Chen HS, Chen SL, Ho YP, Ho KY, Wu YM, et al. Lipid peroxidation: a possible role in the induction and progression of chronic periodontitis. J Periodontal Res 2005;40(5):378–84. 12. Akalin FA, Baltaciog˘lu E, Alver A, Karabulut E. Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis. J Clin Periodontol 2007;34(7):558–65. 13. Tomofuji T, Irie K, Sanbe T. Periodontitis and increase in circulating oxidative stress. Jpn Dent Sci Rev 2009;45(1):46–51. 14. Katsuragi H, Ohtake M, Kurasawa I, Saito K. Intracellular production and extracellular release of oxygen radicals by PMNs and oxidative stress on PMNs during phagocytosis of periodontopathic bacteria. Odontology 2003;91(1):13–8. 15. Sculley DV, Langley-Evans SC. Salivary antioxidants and periodontal disease status. Proc Nutr Soc 2002;61(1):137–43. 16. Massaro M, Habib A, Lubrano L, Del Turco S, Lazzerini G, Bourcier T, et al. The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKC epsilon inhibition. Proc Natl Acad Sci USA 2006;103(41):15184–9. 17. Richard D, Wolf C, Barbe U, Kefi K, Bausero P, Visioli F. Docosahexaenoic acid down-regulates endothelial Nox4 through a sPLA2 signalling pathway. Biochem Biophys Res Commun 2009;389(3):516–22. 18. Arab K, Rossary A, Flourie´ F, Tourneur Y, Steghens JP. Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating gamma-glutamylcysteinyl ligase and glutathione reductase. Br J Nutr 2006;95(1):18–26. 19. Bannenberg GL, Chiang N, Ariel A, Arita M, Tjonahen E, Gotlinger KH, et al. Molecular circuits of resolution: formation and actions of resolvins and protectins. J Immunol 2005;174(7):4345–55. 20. Stillwell W, Wassall SR. Docosahexaenoic acid: membrane properties of a unique fatty acid. Chem Phys Lipids 2003;26(1):1–27. 21. Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients 2010;2:355–74. 22. Salem Jr N, Litman B, Kim HY, Gawrisch K. Mechanism of action of docosahexaenoic acid in the nervous system. Lipids 2001;36(9):945–59. 23. Ziboh VA, Cohen KA, Ellis CN, Miller C, Hamilton TA, Kragballe K, et al. Effects of dietary supplementation of fish oil on neutrophil and epidermal fatty acids Modulation of clinical course of psoriatic subjects. Arch Dermatol 1986;122(11):1277–82. 24. Naqvi AZ, Buettner C, Phillips RS, Davis RB, Mukamal KJ. n-3 fatty acids and periodontitis in US adults. J Am Diet Assoc 2010;110(11):1669–75. 25. Liu X, Zweier JL. A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: evaluation in human polymorphonuclear leukocytes. Free Radic Biol Med 2001;31(7):894–901. 26. Mokhlis GR, Matis BA, Cochran MA, Eckert GJ. A clinical evaluation of carbamide peroxide and hydrogen peroxide whitening agents during daytime use. J Am Dent Assoc 2000;131(9):1269–77. 27. Reis A, Tay LY, Herrera DR, Kossatz S, Loguercio AD. Clinical effects of prolonged application time of an in-office bleaching gel. Oper Dent 2011;36(6):590–6. 28. Zekonis R, Matis BA, Cochran MA, Al Shetri SE, Eckert GJ, Carlson TJ. Clinical evaluation of in-office and at-home bleaching treatments. Oper Dent 2003;28(2):114–21.

archives of oral biology 60 (2015) 144–153

29. Fridovich I. Superoxide anion radical (O2), superoxide dismutases, and related matters. J Biol Chem 1997;272(30):18515–7. 30. Simpson JA, Narita S, Gieseg S, Gebicki S, Gebicki JM, Dean RT. Long-lived reactive species on free-radical-damaged proteins. Biochem J 1992;282(Pt 3):621–4. 31. Gieseg SP, Simpson JA, Charlton TS, Duncan MW, Dean RT. Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins. Biochemistry 1993;32(18):4780–6. 32. Kurita-Ochiai T, Seto S, Suzuki N, Yamamoto M, Otsuka K, Abe K, et al. Butyric acid induces apoptosis in inflamed fibroblasts. J Dent Res 2008;87(1):51–5. 33. Royall JA, Ischiropoulos H, Szejda P, Seeds MC, Thomas M. Evaluation of 20 ,70 -dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 1993;302(2):348–55. 34. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82(1):70–7. 35. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 36. Pendergrass W, Wolf N, Poot M. Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A 2004;61(2):162–9. 37. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226(1):497–509. 38. Teramoto S, Tomita T, Matsui H, Ohga E, Matsuse T, Ouchi Y. Hydrogen peroxide-induced apoptosis and necrosis in human lung fibroblasts: protective roles of glutathione. Jpn J Pharmacol 1999;79(1):33–40. 39. Kurita-Ochiai T, Amano S, Fukushima K, Ochiai K. Cellular events involved in butyric acid-induced T cell apoptosis. J Immunol 2003;171(7):3576–84. 40. Jia Z, Moulson CL, Pei Z, Miner JH, Watkins PA. Fatty acid transport protein 4 is the principal very long chain fatty acyl-CoA synthetase in skin fibroblasts. J Biol Chem 2007;282(28):20573–83. 41. Ibrahimi A, Sfeir Z, Magharaie H, Amri EZ, Grimaldi P, Abumrad NA. Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport. Proc Natl Acad Sci U S A 1996;93(7):2646–51. 42. Kim HY, Akbar M, Lau A, Edsall L. Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3): role of phosphatidylserine in antiapoptotic effect. J Biol Chem 2000;275(45):35215–43523. 43. Akbar M, Calderon F, Wen Z, Kim HY. Docosahexaenoic acid: a positive modulator of Akt signaling in neuron survival. Proc Natl Acad Sci U S A 2005;102(31): 10858–63. 44. Stillwell W, Shaikh SR, Zerouga M, Siddiqui R, Wassall SR. Docosahexaenoic acid affects cell signalling by altering lipid rafts. Reprod Nutr Dev 2005;45(5):559–79. 45. Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. 1966. Biochim Biophys Acta 2011;1807(12):1507–38.

153

46. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, et al. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 1995;15(4):961–73. 47. Vergun O, Keelan J, Khodorov BI, Duchen MR. Glutamateinduced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones. J Physiol 1999;519(Pt 2):451–66. 48. Chicco AJ, Sparagna GC. Role of cardiolipin alterations in mitochondrial dysfunction and disease. Am J Physiol Cell Physiol 2007;292(1):C33–44. 49. Parker MA, King V, Howard KP. Nuclear magnetic resonance study of doxorubicin binding to cardiolipin containing magnetically oriented phospholipid bilayers. Biochim Biophys Acta 2001;1514(2):206–16. 50. Marı´ M, Morales A, Colell A, Garcı´a-Ruiz C, Ferna´ndez-Checa JC. Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 2009;11(11):2685–700. 51. Ott M, Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S. Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci U S A 2002;99(3):1259–63. 52. Raza Shaikh S, Brown DA. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids. Prostaglandins Leukot Essent Fatty Acids 2013;88(1):21–5. 53. Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, et al. Bid Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002;111(3):331–42. 54. Makino N, Mochizuki Y, Bannai S, Sugita Y. Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts. J Biol Chem 1994;269(2):1020–5. 55. Masaki H, Okano Y, Sakurai H. Differential role of catalase and glutathione peroxidase in cultured human fibroblasts under exposure of H2O2 or ultraviolet B light. Arch Dermatol Res 1998;290(3):113–8. 56. Guillot N, Debard C, Calzada C, Vidal H, Lagarde M, Ve´ricel E. Effects of docosahexaenoic acid on some megakaryocytic cell gene expression of some enzymes controlling prostanoid synthesis. Biochem Biophys Res Commun 2008;372(4):924–8. 57. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods 1983;65(1–2):55–63. 58. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 2005;11:127–52. 59. Starkov A, Fiskum G. Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state. J Neurochem 2003;86(5):1101–7. 60. Gutman M. Modulation of mitochondrial succinate dehydrogenase activity, mechanism and function. Mol Cell Biochem 1978;20(1):41–60. 61. Deore GD, Gurav AN, Patil R, Shete AR, Naiktari RS, Inamdar SP. Omega 3 fatty acids as a host modulator in chronic periodontitis patients: a randomised, double-blind, palcebocontrolled, clinical trial. J Periodontal Implant Sci 2014;44(1):25–32.