BPA, superoxide dismutase from bovine erythrocytes, cytochrome C from equine heart, Griess reagent, nitroblue tetrazolium (NBT), N-formyl-Met-Leu-Phe (fMLP), and latex were purchased from Merck Millipore (Burlington, MA). May–Grünwald and Giemsa stains were obtained from Aqua-med. Polimorphoprep™ (Axis Shield) and phosphate-buffered saline (PBS) with or without CaCl2 and MgCl2 ions were supplied by Thermo Fisher Scientific (Waltham, MA). Wortmannin was purchased from Calbiochem (San Diego, CA). ICI 182.780 (ICI/Fulvestrant, 98% pure) and 17β-estradiol (E2) were obtained from Sigma-Aldrich.
The study involved a group of 15 healthy voluntary blood donors [5 women (in the follicular phase of the menstrual cycle) aged 22–25 years and 10 men aged 23–30 years], who were recruited from the Regional Centre for Transfusion Medicine, Bialystok, Poland. All the donors were healthy, and did not smoke or drink alcohol in the last 48 hours before blood collection. The donors also had no history of chronic diseases or immunological deficiencies in the last 5 years. All the donors gave written informed consent before blood donation. For the study, 9 ml of whole blood was taken from a vein in the arm of each donor and transferred to a test tube with anticoagulant (NH Sodium Heparin anticoagulant, Greiner Bio-One GmbH). The human material study was carried out according to the recommendations of the Bioethics Committee of the Medical University of Białystok (Resolution No. R-I-002/396/2019, R-I-002/333/2017). All the experiments were performed in accordance with Good Laboratory Practice.
E2 was used at a physiological concentration of 100 pM. In the earlier work , we found that the mean BPA concentration in the serum samples of healthy people was, respectively, 14.94 nM in women and 17.17 nM in men. Based on this observation, BPA was used at two concentrations in the present study: 16 nM—mean BPA concentration in donor blood; 1.6 µM—a 100-fold higher BPA concentration than the mean concentration in the human serum, which referred to high exposure to this xenoestrogen. Data from the literature suggest that BPA is accumulated in humans from different products (Table 1).
The preliminary cell isolation—separation of polymorphonuclear cells (PMNs) (contains 91% of neutrophils) from the peripheral blood mononuclear cells (contains 94% of lymphocytes)—was carried out by centrifuging the blood samples in density gradient using Polymorphprep™ reagent (AXIS-SHIELD PoC AS, Oslo, Norway). Briefly, the blood was carefully layered on the reagent, the amount of which was equal to the amount of blood used for separation. Subsequently, the sample was centrifuged at 400 g for 30 minutes at room temperature. Cells were counted in the Bürker chamber after staining the nuclei with Türk’s solution. To obtain a pure neutrophil fraction (99.9%) from the PMN fraction, an additional isolation step was carried out using magnetic separation (MACS® Separator) with antibodies and magnetic CD16 MicroBeads (catalog no. 130-045-701, Miltenyi Biotec) . The survival of neutrophils was evaluated using trypan blue under a light microscope, which was found to be 97%.
Assessment of cell capacity of chemotaxis by Boyden chamber
The chemotactic ability of neutrophils was assessed using the Boyden chamber (Neuro Probe, Gaithersburg, MD), a chemoattractant solution (fMLP), and a filter with a pore size of 5 µm diameter. The Boyden chamber consists of two compartments separated by a filter. One of the compartments was filled with fMLP—neutrophils will be attracted to the direction of increasing concentration of chemoattractant. The second compartment was filled with 50000 neutrophils suspended in PBS with CaCl2 and MgCl2 ions. The isolated cells were previously preincubated for 30 minutes in an incubator with 5% CO2 (Nuarie™ US Autoflow, Plymouth, MN) at 37°C without any additional substances, or with BPA (16 nM or 1.6 µM) or E2 (100 pM). After both compartments were filled, the chamber was incubated for an hour in an incubator with 5% CO2 (Nuarie™ US Autoflow) at 37°C. Then, the solutions were removed, and the filter was placed on a slide. The filter was stained using the May–Grünwald–Giemsa staining method and evaluated under a light microscope with oil immersion. All the neutrophils found on the filter and those sticking to the filter pores were counted. The percentage of cells capable of chemotaxis was calculated from the proportion of 50000 cells as 100 %.
Assessment of cell capacity of phagocytosis by Park’s method with latex beads
Blood collected on heparin was centrifuged at 2000 rpm for 5 minutes. The neutrophils collected from the “leukocyte coat” fraction were first incubated in an incubator at 37°C, with a constant flow of 5% CO2 (Nuarie™ US Autoflow), for 30 minutes without or with BPA (16 nM or 1.6 µM) or E2 (100 pM). Latex (exogenous beads) was added to the suspension, which was absorbed into the cytoplasm of cells that were capable of phagocytosis. Then, the suspension was incubated in an incubator with 5% flow of CO2 (Nuarie™ US Autoflow) at 37° temperature for 30 minutes and in at room temperature for the next 30 minutes. After incubation, smears were prepared, fixed, and stained using the May–Grünwald–Giemsa staining method. The smears were evaluated under oil immersion in a light microscope, and the neutrophils were differentiated into four classes based on the amount of the absorbed latex beads: class 0—neutrophils without absorbed latex beads, class I—neutrophils containing 1–10 latex beads, class II—neutrophils containing 11–30 latex beads, and class III—neutrophils containing more than 30 latex beads in the cytoplasm. This allowed the quantitative (percentage of cells capable of phagocytosis) and qualitative (Social Cohesion and Reconciliation (SCORE) index)) assessment of phagocytosis. The SCORE index was obtained by multiplying the group’s name and the number of neutrophils in the group.
Assessment of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity by Park’s test with NBT
The use of Park’s test with NBT enables analyzing the NADPH oxidase activity of human neutrophils. The method is based on the reduction of NBT absorbed into the cell cytoplasm into insoluble blue formazan crystals.
Whole-blood samples were incubated for 30 minutes without or with BPA (16 nM or 1.6 µM) or E2 (100 pM). Then, NBT was added, moreover, latex beads which stimulated cells to absorption were added into NBT stimulated samples. The samples were then incubated for 15 minutes at 37°C with a constant flow of 5% CO2 (Nuarie™ US Autoflow) and 15 minutes at room temperature. Then, smears were prepared on slides, fixed, and stained using the May–Grünwald–Giemsa staining method. The smears were evaluated under oil immersion in a light microscope. The results were expressed as the percentage of cells with formazan in the spontaneous test or as the percentage of cells with formazan and latex in the stimulated test.
Neutrophil extracellular traps
Isolated neutrophils were suspended in a culture medium containing Rosewell Park Memorial Institute 1640, antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml), and serum (4%). They were stimulated to form NETs on 96-well culture plates. For this purpose, the cells were introduced in an amount of 5 × 104 per well. Then, they were incubated for 60 minutes at 37°C under 5% CO2 (Nuarie™ US Autoflow) in the absence or presence of LPS (10 µM), BPA (16 nM or 1.6 µM), or E2 (100 pM). Hoechst 3342 dye (Invitrogen) (at a concentration of 1 µg/ml prepared in PBS), which allows the detection of DNA, and antihuman MPO antibody (Life Technologies), which allows detecting the main NET component, were added to the samples. The analysis of NET formation in the wells was carried out under a fluorescence microscope (IN Cell Analyzer 2200, GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program. The stained cells were counted in nine 20× microscopic fields per section. The results were expressed as the percentage of cells showing the NET formation related to all the neutrophils of an image. For statistical analysis, the mean value of nine images was used for calculating the average values for each sample.
Cluster of differentiation
Isolated neutrophils were suspended in Hank’s Balanced Salt Solution (HBSS) 1X culture medium (5 × 105 cells/ml) in the presence of serum (4%) and antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml). The microplate (Microtest III-Falcon) was incubated for 2 hours at 37°C, with a constant flow of 5% CO2 (Nuarie™ US Autoflow), in the presence of LPS (10 µM), BPA (16 nM or 1.6 µM), or E2 (100 pM). Then, the plate was centrifuged, and after washing and suspending in PBS, the neutrophils were assessed for the expression of CD14 and CD284.
The expression of cluster of differentiation (CD) antigens on the cells was determined by the direct fluorescence method using Canto II flow cytometer (Becton Dickinson) and monoclonal antibodies (Becton Dickinson). Briefly, 20 µl/5 µl of anti-CD14/anti-CD284 monoclonal antibodies, respectively, was added to 50 µl of the neutrophils suspended in PBS. After 30 minutes of incubation in the dark, the cells were resuspended in PBS and analyzed for 30 minutes using the FASCSDiva software. Simultaneously, an isotypic sample was prepared which was used for analyzing the obtained results.
Assessment of total NO concentration in neutrophil supernatants by Griess reaction
Isolated neutrophils were suspended in HBSS 1X culture medium (5 × 105 cells/ml) in the presence of serum (4%) and antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml). The microplate (Microtest III-Falcon) was incubated for 2 hours at 37°C, with a constant flow of 5% CO2 (Nuarie™ US Autoflow), without or with BPA (16 nM or 1.6 µM) or E2 (100 pM). Some cells were previously preincubated for 60 minutes with a wortmannin inhibitor (0.1 µM, Calbiochem, San Diego, CA) or with ICI 182.780 (1 µM, ICI/Fulvestrant, Sigma-Aldrich, 98% pure). Then, the plate was centrifuged and the culture supernatants were used to assess the total NO concentration. The cell precipitate was collected and used for assessing protein expression by Western blot.
NO production by neutrophils was assessed using an indirect method in which the concentration of NO2‑ ions in culture supernatants was measured according to the Griess reaction. The total NO concentration was calculated as the sum of the nitrate (III) and nitrate (V) concentrations. The Griess reaction was used for detecting only nitrates (III) and involved two stages. In the first stage, the neutrophil supernatants were incubated for 30 minutes with cadmium to reduce nitrates (V) to nitrates (III). In the second stage, the Griess reagent was added, and the absorbance was read at 540 nm using a spectrophotometer. The concentration of NO products was expressed in µM (5 × 105 cells in 270 ml of supernatant).
The cytoplasmic fraction of neutrophils was isolated using the NucBuster™ Protein Extraction Kit (Merck). The isolated cytoplasmic protein was suspended in 2X Laemmli Sample Buffer (Bio-Rad) with βME (Bio-Rad). Then, the protein was applied to polyacrylamide gel in an amount of 10 µg/well, and electrophoresis under denaturing conditions (SDS-PAGE) was performed in Mini-PROTEAN® Tetra Cell (Bio-Rad). Using the Transfer Buffer solution, the separated proteins were electrophoretically transferred from the gel to a nitrocellulose membrane on a 0.45 m roll (Bio-Rad) in the Mini-PROTEAN® Tetra Cell apparatus. The next stages of the procedure were carried out in the SNAP i.d.® 2.0 (Millipore) apparatus. The nitrocellulose membrane was blocked in 1X Tris-buffered saline (TBS) with 1% Casein Blocker (Bio-Rad) and incubated for 10 minutes at room temperature with primary antibodies: anti-p-PI3K goat polyclonal antibody, anti-p-Akt1/2/3 (B-5) mouse monoclonal antibody, anti-p-Akt1/2/3 (Ser473) rabbit polyclonal antibody, and anti-iNOS rabbit polyclonal antibody (1:100, Santa Cruz Biotechnology). The membrane was rinsed three times in TBS (Bio-Rad) with Tween® 20 (Sigma). Then, it was incubated for 10 minutes at room temperature with appropriate secondary antibodies labeled with alkaline phosphatase (1:5000, Santa Cruz Biotechnology) and rinsed three times again. Immunoreactive strips were obtained by adding BCIP®/NBT Liquid Substrate System (Sigma). The intensity of the stained strips was evaluated using the ImageJ software (NIMH, Bethesda, MD). The results were expressed in arbitrary units.
All data are reported as mean ± standard deviation (SD). Untransformed variables were statistically analyzed if the following criteria were met: data were normally distributed (under the assumption that SD was less than one-half of the mean of nonnegative variables) and the Shapiro–Wilk W-test was insignificant. For comparison between groups, the t-Student test was used. A significance level of p < 0.05 was considered statistically significant. STATISTICA version 13.3 program (StatSoft, Inc., Tulsa, OK) was used for all analyses.