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Abstract

The aim of the study was to evaluate the effect of bisphenol A (BPA) on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by neutrophils with regard to sex and nuclear factor-κB (NF-κB) pathway participation in this process. This study demonstrated that BPA intensifies the production of NO and the expression of iNOS in the cytoplasmic fraction of neutrophils of women as well as men. In addition, an enhanced expression of NF-κB in the cytoplasmic and nuclear fraction of neutrophils exposed to BPA was observed in the cells of both sexes. The lipopolysaccharide (LPS) stimulation of neutrophils of both sexes led to an intensification of NO production and expression of all tested proteins. However, simultaneous stimulation of neutrophils of both men and women with LPS and BPA decreased the production of NO and expression of iNOS and NF-κB in both fractions compared to the cells exposed only to xenoestrogen. Moreover, expression of iNOS and NF-κB was higher in female neutrophils than in male cells. This study demonstrated that BPA affects the production of NO with the participation of iNOS by human polymorphonuclear neutrophils. This process is associated with the activation of the NF-κB pathway. In addition, different activity of NF-κB in neutrophils, observed with respect to sex, indicates a different role of this pathway in female and male cells.

Keywords

BPA neutrophils nitric oxide inducible nitric oxide synthase NF-κB

Introduction

Every day we are exposed to contact with thousands of harmful chemicals. Many of them are dangerous to our health. Unfortunately, despite the alarming test results, as well as existing replacements, xenoestrogens (“pseudohormones” or “hormonal modulators”) remain in common use. One such compound is bisphenol A (BPA), which is used in the production of plastics, as well as epoxy resins that are used to coat the inner surface of cans for food and beverages packaging. BPA readily penetrates into food and beverages at elevated temperatures or as a result of packaging damage. This compound enters the human body mainly through the digestive tract and through the respiratory system and skin.^1,2 BPA can have harmful effects on human health even at low concentrations. This factor interferes with the hormonal homeostasis of the body, leading to infertility and cancer diseases.^3,4

BPA can interact with the endocrine system and modulate its activity in a manner characteristic of estrogens. This property is obtained through the ability to connect to the estrogen receptor ERα and ERβ as well as to the membrane estrogen receptor.⁵ Changes in the secretion of female sex hormones during the menstrual cycle, pregnancy, and the menopausal period can have a significant effect on the functions of immune system cells.^6

–11 The literature data describe in detail the effect of estrogen and progesterone on the proper functioning of the immune system during bacterial infection, promoting the proinflammatory or anti-inflammatory phenotype of its components.¹² The recognition of the differences in female and male immune systems functioning is the current subject of the research. These discrepancies in large part result from the action of estrogen and progesterone. These hormones can modulate, inter alia, the secretion of proinflammatory cytokines (interleukin IL-1, IL-6, and tumor necrosis factor (TNF-α)) by monocytes, the production of free radicals, and the process of chemotaxis.¹³ Thus, it seems that due to its estrogenic activity BPA may differently affect the activity of the female and male immune systems.

BPA may also affect the cell in an extra-receptor manner, influencing the process of histone protein methylation. It changes the gene expression.¹⁴ Moreover, this xenoestrogen may also cause disorders in the body through oxidative stress. The activity of this xenobiotic leads to a decrease in the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase in mitochondria and liver areas rich in microsomal fraction.^1,15,16

The concentration of biologically active BPA in the serum varies between 0.2 ng/ml and 20 ng/ml. The National Health and Nutrition Examination Survey program detected the presence of BPA in 93% of urine samples. In addition, women had a significantly higher level of this xenoestrogen in the urine in relation to men. The tolerable daily intake is 4 µg/kg body weight/day.^17,18

Despite numerous scientific studies on BPA, its effect on the population of immunocompetent peripheral blood cells, which are neutrophils (polymorphonuclear neutrophils (PMNs)), remains unexplained.^19
–21 These cells are an important element of nonspecific response, and their proper functioning determines the immediate defensive response of the body to the attack of bacteria, fungi, and viruses. They also take part in the removal of improperly functioning or used cell elements and cancer cells.^22,23 Neutrophils effectively eliminate pathogenic factors using, for example, oxygen-dependent killing mechanisms (production of reactive oxygen and nitrogen species, including superoxide anion and nitric oxide (NO)).²⁴ One of the enzymes responsible for NO synthesis is inducible nitric oxide synthase (iNOS), which is controlled by various signaling pathways including MAP, Src, and Akt/PKB kinases. An effect of the signaling cascade activity is an activation of the expression of many genes, like genes encoding transcription factors including nuclear factor-κB (NF-κB), which may regulate the expression of iNOS and the release of NO, inter alia, through the promoter sequence of the iNOS gene.^25
–27

Figure 1.

Concentrations of total NO from PMN. *p < 0.05: significantly different between cells without and with BPA; ^a p < 0.05: cells incubated only with LPS and cells incubated without BPA and LPS; and ^b p < 0.05: cells incubated only with LPS and cells simultaneously incubated with LPS and BPA. BPA: bisphenol A; LPS: lipopolysaccharide; NO: nitric oxide; PMNs: polymorphonuclear neutrophils.

Another mechanism of neutralization and elimination of the pathogenic factor by neutrophils is the oxide-independent killing mechanism, which is associated with the degranulation of serine proteinases: cathepsin G, proteinase 3, and elastase.²⁸ Our previous studies demonstrated the changes in the expression of serine proteases in neutrophils of women and men as a result of BPA activity. Due to the above results, we undertook further study aimed at an evaluation of BPA effect on NO production and iNOS expression by neutrophils considering sex and NF-κB pathway participation in this process.

Due to the strong relationship between the NO concentration and the physiological state of the whole organism, knowledge of signaling pathways and transcription factors regulating iNOS expression may be important to better understand the mechanisms leading to a variety of pathological conditions associated with the formation of many diseases in people exposed to BPA.

Materials and methods

Reagents

BPA and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) liquid substrate system were purchased from Sigma (Steinheim, Germany). Laemmli buffer, Tris-buffered saline (TBS)/Casein buffer, and TBS-T (containing TBS and Tween 20) were purchased from Bio-Rad Laboratories (Hercules, California, USA).

Figure 2.

Expression of iNOS and NF-κB in human PMNs. PMNs were treated for 20 h of BPA (25, 11, and 6.25 ng/μl) and/or LPS (10 µg/ml). The cytoplasmic fractions obtained from the cells were used to detect the expression of the proteins by Western blot. Shown results are representative of five independent experiments. Band intensity was quantified using ImageJ software and expressed in arbitrary units (A.U.). Data shown are mean (± standard error) of five independent experiments. *p < 0.05: significantly different between cells without and with BPA; ^a p < 0.05: cells incubated only with LPS and cells incubated without BPA and LPS; ^b p < 0.05: cells incubated only with LPS and cells simultaneously incubated with LPS and BPA; ^c p < 0.05: cells collected from women and men; and ^d p < 0.05: cells incubated only with BPA and cells incubated with LPS and BPA. BPA: bisphenol A; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; NF-κB: nuclear factor-κB; PMNs: polymorphonuclear neutrophils.

Isolation and incubation of PMNs

The study involved a group of 15 healthy (aged 20–25 years) volunteer blood donors (women [in the first phase of the menstrual cycle] and men), nonsmoking, nonalcoholic, not under drug therapy, and with no recent history of immunological deficiencies. The Ethics Committee of the Medical University of Bialystok (R-I-002/141/2016) approved this study. All participants gave written informed consent prior to blood donations. PMNs were isolated from heparinized (10 U/ml heparin; Polfa, Lodz, Warszawa, Poland) whole blood by density centrifugation using Polymorphprep™ (Axis-Shield PoC AS, Oslo, Norway). This method enables simultaneous separation of two highly purified leukocyte fractions: PMNs (containing 91% PMNs) and peripheral blood mononuclear cell (containing 94% lymphocytes). The purity of isolated PMNs was determined by May–Grunewald–Giemsa. Sera were obtained from blood samples collected without anticoagulation agents.

PMNs were suspended at a concentration of 5 × 10⁶ cells/ml in Hanks’ balanced salt solution (Invitrogen, Carlsbad, California, USA) containing the subject’s own serum (7.4%, 20/270 µl), 100 U penicillin/ml, and 50 ng streptomycin/ml (Polfa Tarchomin SA, Warsaw, Poland). The cells (200 µl aliquots) were then placed into the wells of microplates (Microtest III-Falcon®; BD Biosciences, Bedford, Massachusetts, USA) and incubated for 20 h at 37°C in a 5% CO₂ incubator (Nuaire™ US Autoflow, Plymouth, Minnesota, USA). PMNs in the wells were then treated with 20 µl BPA to attain a final concentration of 25 ng BPA/µl, 11 ng BPA/µl, or 6.25 ng BPA/µl in the well; control wells received vehicle only. Half of the cultured PMNs were incubated in the presence of 10 µg/ml lipopolysaccharide (LPS) and without or with BPA in each concentration. Control wells received vehicle only.

Figure 3.

Expression of iNOS and NF-κB in human PMNs. PMNs were treated for 20 h of BPA (25, 11, and 6.25 ng/μl) and/or LPS (10 µg/ml). The nuclear fractions obtained from the cells were used to detect the expression of the proteins by Western blot. Shown results are representative of five independent experiments. Band intensity was quantified using ImageJ software and expressed in arbitrary units (A.U.). Data shown are mean (± standard error) of five independent experiments. *p < 0.05: significantly different between cells without and with BPA; ^a p < 0.05: cells incubated only with LPS and cells incubated without BPA and LPS; ^b p < 0.05: cells incubated only with LPS and cells simultaneously incubated with LPS and BPA; ^c p < 0.05: cells collected from women and men; and ^d p < 0.05: cells incubated only with BPA and cells incubated with LPS and BPA. BPA: bisphenol A; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; NF-κB: nuclear factor-κB; PMNs: polymorphonuclear neutrophils.

Assay for nitrite production

The synthesis of NO was determined by an assay of the culture supernatant for nitrite, a stable reaction product of NO with molecular oxygen. Total NO concentration is commonly determined as the sum of the nitrite and nitrate concentrations present. NO production by PMNs was determined using an indirect method based on the measurement of nitrite concentration in culture supernatants according to a Griess reaction. In the analyzed samples, nitrate was reduced to nitrate in the presence of cadmium and then converted to nitric acid that yielded a color reaction with Griess reagent. Nitrite concentrations were determined by spectrophotometric analysis at 540 nm with extrapolation from a standard curve prepared in parallel. NO products were expressed in µM (10⁶ cells in 270 µl supernatant).

Protein isolation and Western blot analyses

Cytoplasmic and nuclear extracts from PMNs (3 × 10⁶ cells total/sample) were prepared using NucBuster™ Protein Extraction Kit (EMD Millipore Corp., Billerica, MA, USA). Protein Extraction Kit (Calbiochem®). Stepwise extraction delivered two distinct cellular protein fractions: cytoplasmic and nuclear. The concentration of protein in each was determined with a Qubit™ Protein Assay Kit (Invitrogen). An antibody (Ab) against PARP-1 (1:5000) and an Ab against β-actin (1:100) were used as internal controls within the nuclear and cytoplasmic fractions, respectively.

The extracts were suspended in Laemmli buffer, loaded at 20 µg/well, and then electrophoresed over a 4% stacking and a 10% separating sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. The resolved proteins were electrotransferred onto 0.45-µm pore-size nitrocellulose membranes (Bio-Rad), then blocked with TBS/Casein buffer, washed with TBS-T (TBS with 0.05% Tween-20), and incubated with Qentix™ Western Blot Signal Enhancer (Thermo Fisher Scientific, Rockford, Illinois, USA). The membranes were then incubated for 10 min at room temperature in SNAP (Protein Detection System; Millipore, Billerica, Massachusetts, USA) with 1:100 dilutions of primary monoclonal Ab against iNOS and NF-κB. After washing with 0.1% TBS-T, the membrane was incubated at room temperature with alkaline phosphatase anti-mouse IgG Ab or anti-goat IgG Ab (1:200). Immunoreactive bands were then visualized using BCIP/NBT liquid substrate system; intensities were determined using ImageJ software (Bethesda, Maryland, USA) and expressed in arbitrary units (A.U.).

Statistics

Results were analyzed using Statistica version 13.1 (StatSoft, Inc., Tulsa, Oklahoma, USA). Data distribution normality was determined using the Kolmogorov–Smirnov test. Since data were not normally distributed, for comparison of variations between assayed groups, Mann–Whitney U nonparametric tests were applied to unrelated results. A p-value ≤0.05 was accepted as statistically significant. All data are presented as mean ± standard error.

Results

The exposure of neutrophils in both women and men showed an increased release of NO in the presence of BPA at a concentration of 25 and 6.25 ng/µl. There were no changes in the generation of NO by neutrophils in both sexes exposed to BPA at a concentration of 11 ng/µl.

Stimulation of female and male neutrophils with LPS demonstrated an increased NO production compared with the cells nonstimulated with and nonexposed to BPA.

In the case of concurrent use of the stimulator—LPS and xenoestrogen—BPA (at a concentration of 25, 11, or 6.25 ng/µl) on neutrophils of women, as well as men, a lower generation of NO was observed compared with the cells stimulated only with LPS.

In the case of concurrent application of LPS and BPA to female neutrophils, any changes in NO generation were observed compared with the cells exposed only to BPA.

There were no statistically significant differences in total NO concentrations between neutrophil supernatants of men and women.

Exposure of female PMN to BPA (at a concentration of 25 or 6.25 ng/µl) showed higher expression of iNOS in the cytoplasmic fraction compared with the cells incubated without xenoestrogen. However, after an application of BPA at a concentration of 11 ng/µl, there were no changes in this protein expression in the analyzed cell fraction. Exposure of female neutrophils to BPA (at all concentrations used) increased the expression of NF-κB p65 in the cytoplasmic and nuclear fractions compared with the cells incubated without xenoestrogen.

In contrast to the results obtained in female neutrophils, the exposure of male neutrophils to all BPA concentrations increased the expression of iNOS in the cytoplasmic fraction and NF-κB p65 in the cytoplasmic and nuclear fraction compared with the cells incubated without xenoestrogen.

Incubation of both female and male neutrophils with LPS showed an increase in iNOS expression in the cytoplasmic fraction and NF-κB p65 in both fractions compared with the cells nonstimulated with LPS and xenoestrogen.

In the case of concurrent use of the stimulator—LPS and xenoestrogen—BPA (at a concentration of 25, 11, or 6.25 ng/µl) on neutrophils of women, as well as men, lower iNOS expression was observed in the cytoplasmic fraction compared with LPS-stimulated cells. A decrease in the expression of NF-κB p65 in both fractions was also noted in the cells of both sexes.

In the case of concurrent use of the stimulator-LPS and xenoestrogen-BPA (at a concentration of 25, 11, or 6.25 ng/µl) on neutrophils of women, there were no changes in iNOS expression in the cytoplasmic fraction compared with the cells exposed only to BPA. However, an enhanced expression of NF-κB p65 in the cytoplasmic fraction and a decrease in the nuclear fraction were found in these cells.

In contrast, male neutrophils incubation with LPS and BPA (at all concentrations) led to an increased expression of iNOS and NF-κB p65 in the cytoplasmic fraction compared with the cells exposed only to BPA. In the case of concurrent use of the stimulator-LPS and BPA (25 ng/µl) on neutrophils of this group, lower expression of NF-κB in the nuclear fraction and higher expression for LPS and BPA stimulation (6.5 ng/µl) compared with the cells exposed only to BPA were demonstrated. There were no changes in this factor in the cells exposed to LPS and BPA at a concentration of 11 ng/µl.

In addition, higher expression of iNOS in the cytoplasmic fraction and NF-κB p65 in both fractions was observed in female neutrophils compared with male cells.

Discussion

The results of the study showed for the first time the effect of BPA on the expression of iNOS and the production of NO by neutrophils of women and men. The observed changes in NF-κB expression in the cytoplasmic and nuclear fraction of neutrophils exposed to BPA indicate that this xenobiotic leads to an activation of NF-κB pathway in these cells.

In this study, it is presented that the exposure of female neutrophils to BPA (at a concentration of 25 or 6.25) leads to an increased iNOS expression and NO production. Due to the significantly higher physiological level of estrogens in women in relation to men, it seems that BPA, as a xenoestrogen in women, can initiate a complex mechanism. The literature data confirm the presence of nuclear receptors ERα and ERβ on female neutrophils, while only the ERα was identified in men.

In turn, it was observed in male neutrophils that lower concentrations of BPA lead to higher NO generation and iNOS expression. The obtained results indicate an occurrence of inverse relationship between the applied BPA dose and the obtained biological effect. The results obtained in the cells of men with xenoestrogen can be explained by the few scientific reports that suggest that only chronic exposure to estradiol increases the expression of iNOS in macrophages.²⁹

The consequence of the relationship demonstrated in male cells may be the formation of peroxynitrites (V).^30,31 High estrogen concentration may contribute to an increased production of superoxide anion radical (O₂ ⁻), resulting in the production of toxic peroxynitrites (V) in the reaction with NO. Due to estrogenic activity, BPA probably may also regulate the amount of produced O₂ ⁻. The formed peroxynitrites (V) as a result of these cells exposing to higher doses of BPA may consequently carry the risk of much more serious health effects compared with less toxic NO. These compounds cause protein nitration, lipid peroxidation, enzyme inactivation, induction of apoptosis, and inhibition of cellular respiration in the mitochondria.^32

–36

The results of the presented study showed that LPS stimulation of neutrophils of both sexes leads to an increased NO production and iNOS, as well as NF-κB expression, suggesting a proper development of inflammatory response with these cells’ participation, expressed through an increased generation of NO.

To evaluate the potential impact of BPA on the ongoing inflammatory process, neutrophils of women and men were concurrently stimulated with LPS and xenoestrogen. The presence of BPA in LPS-stimulated neutrophil cultures leads to a decrease in NO concentration and in iNOS expression in cells of both sexes. This situation may be due to the higher affinity of the bacterial toxin to Toll-like receptors (TLRs) compared to BPA. LPS induces TNF-α and NO production on MyD88-dependent and MyD88-independent pathway.³⁷ Xenoestrogens, including BPA, through the pathway independent of MyD88 protein with the participation of IFN-β, inhibit LPS-induced NF-κB activation, leading to the weakening of NO production.³⁸

Thus, it seems that in case of simultaneous exposure of neutrophils to BPA and LPS, xenoestrogen can inhibit the proinflammatory response induced by the bacterial toxin. In turn, the exposure of neutrophils to BPA alone leads to an increased NO production. Therefore, it can be concluded that, depending on the conditions, this xenobiotic can adequately increase or inhibit the production of NO by human neutrophils.

However, it seems surprising in the study presented that the expression of iNOS in female neutrophils was higher than the expression in male PMN despite a similar generation of NO. One of the reasons for the observed differences in iNOS expression between these cells may be the previously mentioned physiologically occurring differences in estrogen concentrations between women and men. Similar observations were made by Molero et al.⁵ who reported the potential causes of comparable NO synthesis in both sexes with a significant activity of neuronal nitric oxide synthase (nNOS) in male cells. They showed that estrogen increases the expression of nNOS present in male neutrophils by interacting only with the nuclear receptor ERα through the involvement of specificity protein-1 and activator protein-1 transcription factors. Accordingly, the similar generation of NO by the cells of both sexes demonstrated in our study may be probably due to the action of BPA, which in male cells led to an activation of nNOS and NO production by neutrophilic ERα. However, in the case of women with physiological high levels of estrogen, BPA could additionally interact with the ERβ, as well as lead to an activation from the TLR4 by activating iNOS with negligible activation of nNOS.

In addition, we also showed significant differences in the activity of NF-κB between neutrophils of women and men. Similar results were obtained by Wilhelm et al.³⁹ who demonstrated that the genes responsible for the expression of NF-κB in women and men are different, indicating a truly dimorphic signaling pattern.

In conclusion, the study demonstrated that BPA affects the production of NO with the participation of iNOS by PMN of women and men. This process is associated with an activation of NF-κB pathway. Moreover, different activity of the NF-κB in neutrophils, observed in relation to sex, indicates a different role of this pathway in female and male cells.

In addition, neutrophils isolated from men are more susceptible to BPA, which results in an increased production of NO at a lower dose compared with women.

Changes in NO production by PMN exposed to BPA may lead to disturbances in the regulation of immune responses controlled by this radical in vivo, for example, in an inflammatory or cancerous process.

Further studies extended to include other neutrophil functions involving signaling pathways and transcription factors will fully explain the molecular mechanism of BPA action on these cells and determine the real evaluation of risk resulting from a wide exposure to this xenoestrogen.

Footnotes

Acknowledgments

The authors are grateful to all the volunteers who participated in this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research was supported by Medical University od Bialystok, Poland (Project Number: N/ST/ZB/15/001/2206).

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Sex-specific differences in the regulation of inducible nitric oxide synthase by bisphenol A in neutrophils