Pesticide Biochemistry and Physiology 118 (2015) 82–89
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p e s t
Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells Dagmara Kurpios-Piec a, Emilia Grosicka-Macia˛g a, Katarzyna Woz´niak b, Cezary Kowalewski b, Ewelina Kiernozek c, Maria Szumiło a, Iwonna Rahden-Staron´ a,* a
Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Department of Dermatology and Immunodermatology, Medical University of Warsaw, Koszykowa 82a, 02-008 Warsaw, Poland c Immunology Department, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland b
A R T I C L E
I N F O
Article history: Received 24 September 2014 Accepted 3 December 2014 Available online 8 December 2014 Keywords: Thiram Cell adhesion molecules (CAMs) Reactive oxygen species NF-kappa B HMEC-1 cells
A B S T R A C T
Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 μg/mL or 2 μg/ mL) (0.2 μM or 0.8 μM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Thiram (tetramethyl thiuram disulfide, TMTD) is a widely used dithiocarbamate pesticide and fungicide as well as an accelerator and vulcanizing agent. TMTD is also known as an inducer of allergic contact dermatitis (ACD) and as an inhibitor of angiogenesis. We have previously demonstrated pro-oxidative effects of thiram in mammalian cells as a decrease in reduced glutathione (GSH), an increase in its oxidized form (GSSG), elevated level of protein oxidation and lipid peroxidation, activation of caspases, and changes in antioxidant enzymes activity [1,2]. These studies suggested that thiram could act through the production of free radicals. Reactive oxygen species (ROS) are important mediators in cell damage, specifically as a factor in endothelial cell damage. A potent
* Corresponding author. Department of Biochemistry, Medical University of Warsaw, 02-097 Warsaw, Banacha 1, Poland. Fax: +48 22 57 20 679. E-mail address: [email protected] (I. Rahden-Staron´). http://dx.doi.org/10.1016/j.pestbp.2014.12.003 0048-3575/© 2014 Elsevier Inc. All rights reserved.
inducer of intracellular ROS formation in endothelial cells is tumor necrosis factor (TNF-α), a pleiotropic inflammatory cytokine [3]. In addition, ROS serve as an intracellular messenger for various redoxsensitive transcription pathways that induce adhesion molecule expression in vascular endothelial cells [4]. TNF-α stimulates and increases the expression of cell adhesion molecules (CAMs) [5], and activates signaling cascades that regulate the activation and translocation of redox-sensitive nuclear transcription factor kappa B (NFκB) [6–8]. Previous studies have shown that NF-κB activation is required for the up-regulation of adhesion molecules such as intercellular cell adhesion molecules (ICAM-1), and vascular cell adhesion molecules (VCAM-1), which are responsible for monocyte adhesion and increased vascular inflammation [9]. The transcription factor NF-κB is sensitive to oxidants, antioxidants, and the conditions affecting the intracellular redox state [10,11]. NF-κB is also associated with cellular functions of proliferation and differentiation [12,13]. Further, it acts as an obligatory mediator of the inflammatory response that causes transcriptional activation of genes encoding, among other proteins, CAMs [6].
D. Kurpios-Piec et al./Pesticide Biochemistry and Physiology 118 (2015) 82–89
CAMs play an important role in immune diseases, inflammation, cell apoptosis, and carcinogenesis [14–16]. Endothelial cells express major adhesion molecules on their surface: intercellular, vascular, and platelet endothelial cell adhesion molecules (PECAM-1) [17]. Endothelial cells are critical components of wound healing, inflammation, circulation, and tumor growth metastases. The immortalized human microvascular endothelial cell line HMEC-1 was used to model the effect of thiram-mediated generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, and PECAM-1. The HMEC-1 cell line was chosen for this work as it provides a reproducible system that has previously been validated to model aspects of the immunobiology of microvascular endothelium, including the uniform response to pro-inflammatory cytokines [18–22]. Thiram shows two opposite effects, that is, oxidative effect due to the presence of disulfide bridge (S-S) in its structure, and reductive effect of the thiol group (-SH) present in dimethyldithiocarbamate anion, which is the product of thiram reduction. Both effects can have an impact on the different cell-mediated responses, including inflammation or/and immunostimulation. In the light of known properties, thiram is considered by Marikovsky and Batya [23] to be used as an inhibitor of angiogenesis and/or inflammation as component of pharmaceutical compositions useful for the treatment of angiogenesis-dependent disorders and/or inflammation associated with a disease. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram or TNF-α alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We demonstrated that human microvascular endothelial cells stimulated in vitro with thiram increase ROS production, activate NF-κB, and increase expression of ICAM-1. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. Expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokines TNF-α alone caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. The exposure of HMEC-1 cells to thiram and TNF-α changed cellular effects compared to the effects of TNF-α alone. We observed further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, elicits in endothelial cell environment effects characteristic for inflammation, and when present concurrently with pro-inflammatory cytokine TNF-α changes the intensity of its action. 2. Materials and methods
83
Invitrogen Life Technologies (Carlsbad, CA, USA). The anti-ICAM-1 (CD54), anti-VCAM-1 (CD106), anti-PECAM (CD31) antibody labeled with PE, PerCp and FITC, respectively, and mouse antibody antihuman NF-κB subunit p65 were purchased from Becton Dickinson (San Diego, CA, USA). Goat anti-mouse FITC conjugated antibody and mouse monoclonal antibody anti-β-actin conjugated with horseradish peroxidase were supplied by Sigma-Aldrich (St Louis, MO, USA). Rabbit polyclonal antibody anti-p65 NF-κB and anti-histone H1 were obtained from Santa Cruz Biotechnology. The secondary goat anti-rabbit IgG peroxidase conjugate antibody was supplied by Merck (Darmstadt, Germany). Fluorescent mounting medium was purchased from Dako (North America, Inc., Carpinteria, CA, USA) and PVDF (polyvinylidene difluoride) membrane from Millipore (Bedford, MA, USA). ECL plus Western Blotting Detection System was purchased from GE Healthcare (Uppsala, Sweden). Cell culture of human microvascular endothelial cells HMEC-1 (ATCC, No CRL-10636) was obtained from American Type Culture Collection (Teddington, UK) and cultured according to its instructions (MCDB 131 medium in a 95% air, 5% CO2 humidified incubator at 37 °C). All media (Gibco BRL) were supplemented with 10% fetal bovine serum, L-glutamine (200 μM), sodium bicarbonate (1.18 g/ L), penicillin/streptomycin and microvascular growth supplement (5%). Cells were treated for 18 h with various concentrations of thiram (0–5 μg/mL) diluted from the stock solution. DMSO (0.1%) was incorporated in all experiments. This concentration of DMSO had no effect on cell growth and was used as solvent control for all tested parameters; all experiments were conducted in triplicate. 2.2. Cell viability assays To measure cell viability, 75–80% confluent cells (8 × 105/6well plate) were treated for 18 hours with thiram (0–5 μg/mL), TNF-α (10 ng/mL) or TNF-α (10 ng/mL) with thiram (0–5 μg/mL). Cells were harvested and cell viability assessed by the trypan blue exclusion assay. Untreated cells were used as the 100% viability value. Cells were counted using Bürker chamber under light microscope. All experiments were conducted in triplicate. The growth inhibition effect of thiram, TNF-α or TNF-α with thiram on HMEC-1 cells was determined by measuring the MTT dye absorbance in cells. MTT is converted to an insoluble formazon by mitochondrial dehydrogenase of living cells. Briefly, cells (2 × 104/ well) were dispensed into 96-well plates in medium (200 μl) and thiram (0–5 μg/mL), TNF-α (10 ng/mL) or TNF-α (10 ng/mL) with thiram (0–5 μg/mL) was added for 18 h at 37 °C. Then, 20 μl MTT (5 mg/mL stock solution) was added to each well. After 4 hours, the reaction was terminated and the plates were incubated to solubilize the formazon dye by adding 200 μl DMSO: isopropanol (1:1). The optical density was measured with an UVM 340 (ASYS Hitech GmbH, Austria) microplate reader at 570 nm. The experiments were conducted in triplicate.
2.1. Compounds and cell exposure 2.3. ROS detection Thiram (tetramethyl thiuram disulfides, CAS 137-26-8), >99% purity, was obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). DMSO (99.9% purity), sodium dodecyl sulfate (SDS), Trypan blue (TB), Thiazolyl blue tatrazolium bromide (MTT), tumor necrosis factor α (TNF-α), non-enzymatic cell dissociation solution and all other general laboratory chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Trypsin-EDTA solution and phosphate buffered saline (PBS) attachment factors were supplied by Gibco BRL. Microvascular growth supplement (MVGS) was purchased from Cascade Biologics (Portland, OR, USA). Cell Wash Buffer and all cell culture plastics were purchased from Becton Dickinson (San Diego, CA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (chloromethyl-DCF-DA), and hydroethidine (HE) were purchased from
HMEC-1 were seeded onto 96-well plates (5 × 104/well) and allowed to adhere for 24 hours. Then cells were rinsed with PBS and incubated with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) or 5 μM hydroethidine (HE) for 30 minutes at 37 °C in the dark. Thereafter, cells were rinsed with PBS, treated with phenol free culture medium containing thiram in concentration 0.5 μg/ mL or 2 μg/mL, and to observe short-living ROS, were incubated for 1 and 3 hours at 37 °C. A sample with 1.5 mM H2O2 was a positive control and a sample without any reagent was a negative control. The generation of H2O2 or O2•− was measured and expressed as fluorescence intensity (FI) by Microplate Spectrofluorometer BioTek Synergy™ (BioTek Instruments, USA). H2DCFDA is oxidized by
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generated H2O2 to 2′,7′-dichlorofluorescein (DCF) emitting fluorescence at 527 nm in response to the 492 nm excitation. In the presence of O2•− HE converts to ethidium bromide. The intensity of its fluorescence was measured using excitation wavelength 510 nm and emission wavelength 590 nm. The fluorescence from three independent experiments was analyzed. 2.4. Endothelial cell NF-κB response to thiram and TNF-α. Indirect analysis of NF-κB induction with laser scanning confocal microscope (LSCM) HMEC-1 cells (6 × 104/well) were grown on glass cover slips in 6-well plates. After cells reached 60–70% confluence, they were incubated in complete medium with thiram (0.5, 2 μg/mL) and/or with 10 ng/mL TNF-α for 1 h. Thereafter, the endothelial cells were washed two times with cold PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 20 min. Then, cells were washed three times with cold PBS, treated with 3% bovine serum albumin (BSA) in PBS containing 0.05% Tween at night at 4 °C and then incubated with mouse antibody anti-human NF-κB subunit p65 (1:350) overnight at 4 °C. After three washes with PBS/3% BSA, goat anti-mouse FITC conjugated antibody (1:150) was applied to the cells for 60 min at room temperature. After washing with PBS, coverslips were mounted with fluorescent mounting medium and analyzed with a Radiance 2000 confocal microscope equipped with a 40-x glycerol immersive objective. The sections were assessed using laser line of 488 nm for FITC and filter 500–560 nm. On average, five to six slides from four independent experiments were examined by LSCM with similar results, and representative experiments are shown in the corresponding photographs.
Fig. 1. Cell viability of thiram treated HMEC-1 cells using TB exclusion assay and MTT test. Values are expressed as percentages of viable cells with respect to controls. All data represent the means ± SD of three experiments, each of them performed in triplicate. aP < 0.001 versus control cells, bP < 0.05 versus control cells.
2.5. Immunoblotting analysis For Western blot analysis of NFkB/p65 subunit protein, nuclear and cytoplasm extracts were obtained according to procedure described in NE-PER® Nuclear and Cytoplasm Extraction Reagents (Pierce, Thermo Scientific; Rockford, IL, USA) and 10 μg protein per sample was separated by 10% SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane by electroblotting. The membrane was blocked in 10% skim milk for 1 h, washed with 3% skim milk in TBS-T (TBS containing 0.05% Tween 20) and probed with antibodies against NF-kB/p65 and H1 (1:500 dilution), and against β-actin (1:4000 dilution) overnight at 4 °C. After extensive washing with 3% skim milk in TBS-T the secondary goat antirabbit IgG was added to p65 and H1 (1:2000 dilution) for 60 min at room temperature. Exposure was carried out with chemiluminescent immunodetection system (ECL plus Western Blotting Detection System). Image Analysis System UVI-KS4000 Syngen Biotech, Scion Co. (Frederick, MD, USA) was used to determine Western blot band density. Assays were repeated three times with similar results, and representative experiments are shown in the corresponding figures. 2.6. Flow cytometry analysis Cell surface expression of adhesion molecules on HMEC-1 cells was analyzed by flow cytometry. After cells (8 × 104/well) reached 80% confluence in 6-well plates, they were incubated for 18 hours in complete medium with thiram (0.5, 2 μg/mL) or TNF-α (10 ng/ mL) alone, or with TNF-α (10 ng/mL) and thiram (0.5, 2 μg/mL). Viability of endothelial cells was unaffected during the time course of these experiments, as assessed by the trypan blue staining. After completion of the treatment, cells were washed with Wash buffer, carefully lifted from culture plates using non-enzymatic cell dissociation solution. Cells were stained with monoclonal antibodies
Fig. 2. Level of reactive oxygen species in HMEC-1 cells stimulated with thiram. (a) Fluorescence intensity of the probe DCF (5 μM), (b) fluorescence intensity of the probe HE (5 μM) in the presence of thiram (0.5 μg/ml or 2 μg/ml) for 1 or 3 h. All data represent the means ± SD of three experiments, each of them performed in triplicate. aP < 0.02 versus control cells, bP < 0.05 versus control cells.
D. Kurpios-Piec et al./Pesticide Biochemistry and Physiology 118 (2015) 82–89
against ICAM-1/PE, VCAM-1/PerCP, and PECAM/FITC according to the standard procedure provided by the company. Three-color analysis was performed on FACSCalibur (Becton-Dickinson, San Jose, CA, USA). Results of four independent experiments are presented as the percentage of positively stained cells. The expression of adhesion molecules was determined by the mean fluorescence intensity (MFI). Analysis was performed using the CellQuest program. Cells stimulated by TNF-α were used as a positive control. 2.7. Statistical analysis All data were representative of experiments done in triplicate and were expressed as the mean ± standard deviation (±SD). The assessment of differences between groups was analyzed by Student’s t-test. The Kolmogorov–Smirnov statistic was used to evaluate changes in the expression of surface markers. Representative histogram is presented. Statistically significant differences between the experimental and control groups are denoted in the figures. Differences were considered significant if the probability (P)-value was
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p e s t
Thiram activates NF-kappaB and enhances ICAM-1 expression in human microvascular endothelial HMEC-1 cells Dagmara Kurpios-Piec a, Emilia Grosicka-Macia˛g a, Katarzyna Woz´niak b, Cezary Kowalewski b, Ewelina Kiernozek c, Maria Szumiło a, Iwonna Rahden-Staron´ a,* a
Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland Department of Dermatology and Immunodermatology, Medical University of Warsaw, Koszykowa 82a, 02-008 Warsaw, Poland c Immunology Department, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland b
A R T I C L E
I N F O
Article history: Received 24 September 2014 Accepted 3 December 2014 Available online 8 December 2014 Keywords: Thiram Cell adhesion molecules (CAMs) Reactive oxygen species NF-kappa B HMEC-1 cells
A B S T R A C T
Thiram (TMTD) is a fungicidal and bactericidal agent used as antiseptic, seed disinfectant and animal repellent. In the light of known properties, thiram is considered to be used as an inhibitor of angiogenesis and/or inflammation. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram at low concentration (0.5 μg/mL or 2 μg/ mL) (0.2 μM or 0.8 μM) or TNF-α (10 ng/mL) alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We carried out an in vitro study aimed at assessing generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, PECAM-1. It was found that TMTD produced ROS and activated NF-κB. Activation of NF-κB was concurrent with an increase in ICAM-1 expression on the surface of HMEC-1 cells. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. The expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was also found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokine TNF-α caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. Treatment with thiram and TNF-α changed cellular response compared with effects observed after treatment with TNF-α alone, i.e. further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, and elicits in endothelial cell environment effects characteristic for inflammation. However, when it is present concurrently with pro-inflammatory cytokine TNF-α interferes with its action. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Thiram (tetramethyl thiuram disulfide, TMTD) is a widely used dithiocarbamate pesticide and fungicide as well as an accelerator and vulcanizing agent. TMTD is also known as an inducer of allergic contact dermatitis (ACD) and as an inhibitor of angiogenesis. We have previously demonstrated pro-oxidative effects of thiram in mammalian cells as a decrease in reduced glutathione (GSH), an increase in its oxidized form (GSSG), elevated level of protein oxidation and lipid peroxidation, activation of caspases, and changes in antioxidant enzymes activity [1,2]. These studies suggested that thiram could act through the production of free radicals. Reactive oxygen species (ROS) are important mediators in cell damage, specifically as a factor in endothelial cell damage. A potent
* Corresponding author. Department of Biochemistry, Medical University of Warsaw, 02-097 Warsaw, Banacha 1, Poland. Fax: +48 22 57 20 679. E-mail address: [email protected] (I. Rahden-Staron´). http://dx.doi.org/10.1016/j.pestbp.2014.12.003 0048-3575/© 2014 Elsevier Inc. All rights reserved.
inducer of intracellular ROS formation in endothelial cells is tumor necrosis factor (TNF-α), a pleiotropic inflammatory cytokine [3]. In addition, ROS serve as an intracellular messenger for various redoxsensitive transcription pathways that induce adhesion molecule expression in vascular endothelial cells [4]. TNF-α stimulates and increases the expression of cell adhesion molecules (CAMs) [5], and activates signaling cascades that regulate the activation and translocation of redox-sensitive nuclear transcription factor kappa B (NFκB) [6–8]. Previous studies have shown that NF-κB activation is required for the up-regulation of adhesion molecules such as intercellular cell adhesion molecules (ICAM-1), and vascular cell adhesion molecules (VCAM-1), which are responsible for monocyte adhesion and increased vascular inflammation [9]. The transcription factor NF-κB is sensitive to oxidants, antioxidants, and the conditions affecting the intracellular redox state [10,11]. NF-κB is also associated with cellular functions of proliferation and differentiation [12,13]. Further, it acts as an obligatory mediator of the inflammatory response that causes transcriptional activation of genes encoding, among other proteins, CAMs [6].
D. Kurpios-Piec et al./Pesticide Biochemistry and Physiology 118 (2015) 82–89
CAMs play an important role in immune diseases, inflammation, cell apoptosis, and carcinogenesis [14–16]. Endothelial cells express major adhesion molecules on their surface: intercellular, vascular, and platelet endothelial cell adhesion molecules (PECAM-1) [17]. Endothelial cells are critical components of wound healing, inflammation, circulation, and tumor growth metastases. The immortalized human microvascular endothelial cell line HMEC-1 was used to model the effect of thiram-mediated generation of reactive oxygen species (ROS), activation of NF-κB, and expression of cell adhesion molecules ICAM-1, VCAM-1, and PECAM-1. The HMEC-1 cell line was chosen for this work as it provides a reproducible system that has previously been validated to model aspects of the immunobiology of microvascular endothelium, including the uniform response to pro-inflammatory cytokines [18–22]. Thiram shows two opposite effects, that is, oxidative effect due to the presence of disulfide bridge (S-S) in its structure, and reductive effect of the thiol group (-SH) present in dimethyldithiocarbamate anion, which is the product of thiram reduction. Both effects can have an impact on the different cell-mediated responses, including inflammation or/and immunostimulation. In the light of known properties, thiram is considered by Marikovsky and Batya [23] to be used as an inhibitor of angiogenesis and/or inflammation as component of pharmaceutical compositions useful for the treatment of angiogenesis-dependent disorders and/or inflammation associated with a disease. Since angiogenesis requires the growth of vascular endothelial cells we have used microvascular endothelial cell line HMEC-1 to elucidate the effect of thiram on normal and stimulated cells. We cultured HMEC-1 cells in the presence of thiram or TNF-α alone, and thiram together with TNF-α. TNF-α was used as a cytokine that triggers changes characteristic for inflammatory state of the cell. We demonstrated that human microvascular endothelial cells stimulated in vitro with thiram increase ROS production, activate NF-κB, and increase expression of ICAM-1. ICAM-1 reflects intensity of inflammation in endothelial cell milieu. Expression of VCAM-1 and PECAM-1 on these cells was not changed by thiram. It was found that stimulation of the HMEC-1 cells with the pro-inflammatory cytokines TNF-α alone caused activation of ICAM-1 and VCAM-1 expression with concomitant decrease of PECAM-1 cell surface expression above the control levels. The exposure of HMEC-1 cells to thiram and TNF-α changed cellular effects compared to the effects of TNF-α alone. We observed further increase of ICAM-1 expression and impairment of the TNF-α effect on PECAM-1 and VCAM-1 expression. This study demonstrated that thiram acts as a pro-oxidant, elicits in endothelial cell environment effects characteristic for inflammation, and when present concurrently with pro-inflammatory cytokine TNF-α changes the intensity of its action. 2. Materials and methods
83
Invitrogen Life Technologies (Carlsbad, CA, USA). The anti-ICAM-1 (CD54), anti-VCAM-1 (CD106), anti-PECAM (CD31) antibody labeled with PE, PerCp and FITC, respectively, and mouse antibody antihuman NF-κB subunit p65 were purchased from Becton Dickinson (San Diego, CA, USA). Goat anti-mouse FITC conjugated antibody and mouse monoclonal antibody anti-β-actin conjugated with horseradish peroxidase were supplied by Sigma-Aldrich (St Louis, MO, USA). Rabbit polyclonal antibody anti-p65 NF-κB and anti-histone H1 were obtained from Santa Cruz Biotechnology. The secondary goat anti-rabbit IgG peroxidase conjugate antibody was supplied by Merck (Darmstadt, Germany). Fluorescent mounting medium was purchased from Dako (North America, Inc., Carpinteria, CA, USA) and PVDF (polyvinylidene difluoride) membrane from Millipore (Bedford, MA, USA). ECL plus Western Blotting Detection System was purchased from GE Healthcare (Uppsala, Sweden). Cell culture of human microvascular endothelial cells HMEC-1 (ATCC, No CRL-10636) was obtained from American Type Culture Collection (Teddington, UK) and cultured according to its instructions (MCDB 131 medium in a 95% air, 5% CO2 humidified incubator at 37 °C). All media (Gibco BRL) were supplemented with 10% fetal bovine serum, L-glutamine (200 μM), sodium bicarbonate (1.18 g/ L), penicillin/streptomycin and microvascular growth supplement (5%). Cells were treated for 18 h with various concentrations of thiram (0–5 μg/mL) diluted from the stock solution. DMSO (0.1%) was incorporated in all experiments. This concentration of DMSO had no effect on cell growth and was used as solvent control for all tested parameters; all experiments were conducted in triplicate. 2.2. Cell viability assays To measure cell viability, 75–80% confluent cells (8 × 105/6well plate) were treated for 18 hours with thiram (0–5 μg/mL), TNF-α (10 ng/mL) or TNF-α (10 ng/mL) with thiram (0–5 μg/mL). Cells were harvested and cell viability assessed by the trypan blue exclusion assay. Untreated cells were used as the 100% viability value. Cells were counted using Bürker chamber under light microscope. All experiments were conducted in triplicate. The growth inhibition effect of thiram, TNF-α or TNF-α with thiram on HMEC-1 cells was determined by measuring the MTT dye absorbance in cells. MTT is converted to an insoluble formazon by mitochondrial dehydrogenase of living cells. Briefly, cells (2 × 104/ well) were dispensed into 96-well plates in medium (200 μl) and thiram (0–5 μg/mL), TNF-α (10 ng/mL) or TNF-α (10 ng/mL) with thiram (0–5 μg/mL) was added for 18 h at 37 °C. Then, 20 μl MTT (5 mg/mL stock solution) was added to each well. After 4 hours, the reaction was terminated and the plates were incubated to solubilize the formazon dye by adding 200 μl DMSO: isopropanol (1:1). The optical density was measured with an UVM 340 (ASYS Hitech GmbH, Austria) microplate reader at 570 nm. The experiments were conducted in triplicate.
2.1. Compounds and cell exposure 2.3. ROS detection Thiram (tetramethyl thiuram disulfides, CAS 137-26-8), >99% purity, was obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). DMSO (99.9% purity), sodium dodecyl sulfate (SDS), Trypan blue (TB), Thiazolyl blue tatrazolium bromide (MTT), tumor necrosis factor α (TNF-α), non-enzymatic cell dissociation solution and all other general laboratory chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Trypsin-EDTA solution and phosphate buffered saline (PBS) attachment factors were supplied by Gibco BRL. Microvascular growth supplement (MVGS) was purchased from Cascade Biologics (Portland, OR, USA). Cell Wash Buffer and all cell culture plastics were purchased from Becton Dickinson (San Diego, CA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (chloromethyl-DCF-DA), and hydroethidine (HE) were purchased from
HMEC-1 were seeded onto 96-well plates (5 × 104/well) and allowed to adhere for 24 hours. Then cells were rinsed with PBS and incubated with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) or 5 μM hydroethidine (HE) for 30 minutes at 37 °C in the dark. Thereafter, cells were rinsed with PBS, treated with phenol free culture medium containing thiram in concentration 0.5 μg/ mL or 2 μg/mL, and to observe short-living ROS, were incubated for 1 and 3 hours at 37 °C. A sample with 1.5 mM H2O2 was a positive control and a sample without any reagent was a negative control. The generation of H2O2 or O2•− was measured and expressed as fluorescence intensity (FI) by Microplate Spectrofluorometer BioTek Synergy™ (BioTek Instruments, USA). H2DCFDA is oxidized by
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D. Kurpios-Piec et al./Pesticide Biochemistry and Physiology 118 (2015) 82–89
generated H2O2 to 2′,7′-dichlorofluorescein (DCF) emitting fluorescence at 527 nm in response to the 492 nm excitation. In the presence of O2•− HE converts to ethidium bromide. The intensity of its fluorescence was measured using excitation wavelength 510 nm and emission wavelength 590 nm. The fluorescence from three independent experiments was analyzed. 2.4. Endothelial cell NF-κB response to thiram and TNF-α. Indirect analysis of NF-κB induction with laser scanning confocal microscope (LSCM) HMEC-1 cells (6 × 104/well) were grown on glass cover slips in 6-well plates. After cells reached 60–70% confluence, they were incubated in complete medium with thiram (0.5, 2 μg/mL) and/or with 10 ng/mL TNF-α for 1 h. Thereafter, the endothelial cells were washed two times with cold PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 20 min. Then, cells were washed three times with cold PBS, treated with 3% bovine serum albumin (BSA) in PBS containing 0.05% Tween at night at 4 °C and then incubated with mouse antibody anti-human NF-κB subunit p65 (1:350) overnight at 4 °C. After three washes with PBS/3% BSA, goat anti-mouse FITC conjugated antibody (1:150) was applied to the cells for 60 min at room temperature. After washing with PBS, coverslips were mounted with fluorescent mounting medium and analyzed with a Radiance 2000 confocal microscope equipped with a 40-x glycerol immersive objective. The sections were assessed using laser line of 488 nm for FITC and filter 500–560 nm. On average, five to six slides from four independent experiments were examined by LSCM with similar results, and representative experiments are shown in the corresponding photographs.
Fig. 1. Cell viability of thiram treated HMEC-1 cells using TB exclusion assay and MTT test. Values are expressed as percentages of viable cells with respect to controls. All data represent the means ± SD of three experiments, each of them performed in triplicate. aP < 0.001 versus control cells, bP < 0.05 versus control cells.
2.5. Immunoblotting analysis For Western blot analysis of NFkB/p65 subunit protein, nuclear and cytoplasm extracts were obtained according to procedure described in NE-PER® Nuclear and Cytoplasm Extraction Reagents (Pierce, Thermo Scientific; Rockford, IL, USA) and 10 μg protein per sample was separated by 10% SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane by electroblotting. The membrane was blocked in 10% skim milk for 1 h, washed with 3% skim milk in TBS-T (TBS containing 0.05% Tween 20) and probed with antibodies against NF-kB/p65 and H1 (1:500 dilution), and against β-actin (1:4000 dilution) overnight at 4 °C. After extensive washing with 3% skim milk in TBS-T the secondary goat antirabbit IgG was added to p65 and H1 (1:2000 dilution) for 60 min at room temperature. Exposure was carried out with chemiluminescent immunodetection system (ECL plus Western Blotting Detection System). Image Analysis System UVI-KS4000 Syngen Biotech, Scion Co. (Frederick, MD, USA) was used to determine Western blot band density. Assays were repeated three times with similar results, and representative experiments are shown in the corresponding figures. 2.6. Flow cytometry analysis Cell surface expression of adhesion molecules on HMEC-1 cells was analyzed by flow cytometry. After cells (8 × 104/well) reached 80% confluence in 6-well plates, they were incubated for 18 hours in complete medium with thiram (0.5, 2 μg/mL) or TNF-α (10 ng/ mL) alone, or with TNF-α (10 ng/mL) and thiram (0.5, 2 μg/mL). Viability of endothelial cells was unaffected during the time course of these experiments, as assessed by the trypan blue staining. After completion of the treatment, cells were washed with Wash buffer, carefully lifted from culture plates using non-enzymatic cell dissociation solution. Cells were stained with monoclonal antibodies
Fig. 2. Level of reactive oxygen species in HMEC-1 cells stimulated with thiram. (a) Fluorescence intensity of the probe DCF (5 μM), (b) fluorescence intensity of the probe HE (5 μM) in the presence of thiram (0.5 μg/ml or 2 μg/ml) for 1 or 3 h. All data represent the means ± SD of three experiments, each of them performed in triplicate. aP < 0.02 versus control cells, bP < 0.05 versus control cells.
D. Kurpios-Piec et al./Pesticide Biochemistry and Physiology 118 (2015) 82–89
against ICAM-1/PE, VCAM-1/PerCP, and PECAM/FITC according to the standard procedure provided by the company. Three-color analysis was performed on FACSCalibur (Becton-Dickinson, San Jose, CA, USA). Results of four independent experiments are presented as the percentage of positively stained cells. The expression of adhesion molecules was determined by the mean fluorescence intensity (MFI). Analysis was performed using the CellQuest program. Cells stimulated by TNF-α were used as a positive control. 2.7. Statistical analysis All data were representative of experiments done in triplicate and were expressed as the mean ± standard deviation (±SD). The assessment of differences between groups was analyzed by Student’s t-test. The Kolmogorov–Smirnov statistic was used to evaluate changes in the expression of surface markers. Representative histogram is presented. Statistically significant differences between the experimental and control groups are denoted in the figures. Differences were considered significant if the probability (P)-value was
