- Open Access
Effects of subchronic exposure of nonylphenol on the expression of immune-related factors and estrogen receptors in the spleen of rats
Environmental Sciences Europe volume 34, Article number: 30 (2022)
Previous studies have shown that EDCs may activate nuclear transcription factor, such as activator protein-1 (AP-1), nuclear factor of activated Tcells (NF-AT) and nuclear factor kappa B (NF-κB) in the process of immune damage. At the same time, some experts believed that estrogen may play an important role in this process. As a typical representative of EDCs, nonylphenol (NP) has not been reported. The aim of this work was to explore the relationship between the immune inflammatory damage and the changes in estrogen expression in male rats during the chronic exposure to NP at environmental concentrations. Sixty SPF Sprague–Dawley rats were divided into five groups (n = 12 per group): blank control group (corn oil), low-dose NP exposure group (0.4 mg/kg/d), medium-dose NP exposure group (4 mg/kg/d), high-dose NP exposure group (40 mg/kg/d), and estradiol control group (E2: 30 μg/kg/d).
Compared with the control group, rat spleen organ coefficient, number of spleen nodules, relative area of lymph nodes and white pulp were relatively reduced in the L (NP, 0.4 mg/kg) and H (NP, 40 mg/kg) exposure dose groups (P < 0.001). Lymphocytes were rich in cytoplasm, mitochondria were swollen, part of the cristae was reduced, and rough endoplasmic reticulum was expanded. The serum levels of IgG (P < 0.001) and IgM (P = 0.002) showed a downward trend. The percentage of Th cells (CD3+CD4+) was significantly decreased (P < 0.001), and the percentage of B lymphocytes shows an opposite trend (P < 0.001). Giemsa staining showed that the number of neutrophils (P < 0.001) was increased. The expressions of estrogen receptor ER-α and ER-β protein in the spleen increased significantly (P < 0.001). The expressions of AP-1 protein and NF-AT protein in the spleen were increased, and the expression of NF-KB protein was decreased (P < 0.001). The expressions of IL-4, ER-α and ER-β (P < 0.001) levels in serum increased. The mRNA-seq bioinformatics detection showed the final differentially expressed immune-inflammatory-related genes between the control and H-NP groups as follow: down-regulated: TLR4, Gata3, IL12, up-regulated: TNF-a, IL10, INOS. The mRNA expressions of ER-α, ER-β, NF-KB, IL4, AP-1, TLR4, Gata3, and NF-AT were consistent with the results of mRNA-seq analysis. NP content was correlated with the expressions of ER-α, ER-β, IL4, AP-1, NF-AT, TLR4, NF-KB, as well as IL-12 proteins in the spleen tissue ([r] < 1, P < 0.05).
Chronic exposure to NP at environmental concentration could cause immune dysfunction, resulting in immunotoxicity and inflammatory effects, and lead to changes in the activity of transcription factors and differential immune inflammatory factors in rats.
As a typical representative of EDCs, nonylphenol (NP) is one of the main metabolites of nonylphenol polyoxyethylene ethers (NPEs), and it is extremely difficult to degrade in the environment. As technical syntheses are based on nonene obtained from trimerization of propene, NP consists of a very complex mixture of isomers with differently branched nonyl groups, and the isomer composition varies depending on the manufacturer. Furthermore, in the environment, isomer specific degradation alters the isomer mixture again. This results in a very complex contamination problem concerning the endocrine-disrupting potential of NP. Theoretically, there are 211 possible constitutional isomers and approximately 100 have been observed in environmentally relevant matrices [1,2,3,4,5,6,7,8,9,10]. A large number of studies have confirmed that EDCs have estrogen effects, can competitively bind to estrogen receptors, and affect the metabolism of estrogen in the body. One of its main mechanisms of function is the interaction with steroid hormone receptors (such as androgens, estrogen, and adrenal hormones), and environmental estrogens are currently the most concerned sex hormones [11,12,13,14,15,16,17,18,19,20]. It can regulate the activity of various immune cells such as T cells, B cells, and macrophages by reducing the synthesis and secretion of various cytokines and immunoglobulins [21, 22].
The structural expression, functional regulation and variation of ER-type receptors in different target tissues and their relationship with the occurrence and development of diseases have been research hotspots in recent years [23,24,25,26]. Humans show strong gender differences in infection and autoimmunity . Experts speculated that sex hormones may regulate immune responses [17, 28]. Molero et al. proved that the expression of ERs in neutrophils depended on the woman’s menstrual cycle . A recent study showed that the sex-specificity of BPA exposure changes the microanatomical structure of spleen cells in CD1 mice . ERα was found to be sensitive to 17β-estradiol in the ER of male neutrophils, confirming that xenoestrogens, as endogenous estrogen, exhibited different effects that may depend on gender . The phenotypic differences in EDCs exposure between men and women may be related to the impact of gender on immune function . Bisphenol A (BPA) has been shown to change the expression of estrogen receptors in terms of sex and dosage. Recently, it has been discovered that BPA may change the function of T cells by regulating the expression of ERs . The inhibition of Th1 cells and the enhancement of Th2 cells caused by NP exposure may be due to the effect of ERs [34, 35]. These results indicated that the estrogen receptor may be a potential target of EDCs immunomodulation [11, 14, 36,37,38,39,40,41].
Nuclear factor of activated Tcells (NF-AT), Activator protein-1 (AP-1), and Nuclear Factor Kappa B (NF-κB) in T cells are multi-functional and important nuclear transcription factors involved in inflammation, immune response and stress-related multiple gene expression. These nuclear transcription factors’ abnormal activation or complete inhibition is related to the occurrence of many diseases [42,43,44]. The regulation of IL-4 by Th cells of bis (2-ethylhexyl) phthalate (DEHP)—exposed mice depended on the activation of the NF-AT of activated T cells [45,46,47]. EDCs blocked the MAPK and NF-κB pathways while activated the NF-AT . Lee et al. found that 4-Octylphenol (4-OP) promoted the production of T cells by activating NF-AT, thereby enhancing the amount of IL-4 . Prenatal exposure of mice to EDCs interfered with the regulation of estrogen receptors, NF-AT, AP-1, and NF-κB nuclear factors, affected Fas/Fas gene ligands, and the differentiation of T cells, and has a significant effect on immune function in the long term .
Exposure to EDCs may cause damage to the body's immune function [51,52,53,54]. Related domestic and foreign studies have shown that EDCs such as BPA and PAE may cause changes of the AP-1, NF-AT, and NF-κB in the process of immune damage. A large amount of data in the literature confirmed the effect of EDCs on the estrogen nuclear receptors in immune cells [55,56,57]. At the same time, some experts believed that estrogen may play an important role in this process . As a typical representative of EDCs, NP has estrogen-like activity [59, 60]. There are few reports on the changes of transcription factors and estrogen during the process of NP-induced immune function damage [42, 47, 58, 61,62,63,64]. In this study, the main route of exposure to NP was simulated by gavage administration through animal experiments, and sub-chronic long-term low-concentration NP exposure was carried out. To investigate whether long-term exposure to low concentrations of NP affects immune function, and the changes and associations between immune factors and estrogen receptors. At the same time, combined with high-throughput screening technology, the overall effect was evaluated for their toxicological effects and relationships, and the possible mechanism model of estrogen in the process of NP acting on immune factors was conceived. To explore the association effects of NP subchronic exposure on estrogen expression, transcription factor changes, immune injury and inflammation in rats.
Materials and methods
Nonylphenol (99% purity, Product Code: BT5655) was purchased from the Shandong Xiya Chemical Industry Co., Ltd. (Shandong, China). Estradiol (E2) was purchased from Zhejiang Lianshuo Biotechnology Co., Ltd. (96.3% purity, Product Code: 2RR9-37EB), the antibody of lymphocyte population detected by flow cytometry was purchased from BD Company (Becton Dickinson, US): CD3 antibody (Product Code: 554833), CD161a antibody (Product Code: 565413), CD45R antibody (Product Code: 561876), CD4 antibody (Product Code: 561833), CD8a antibody (Product Code: 561611). Hematoxylin and Eosin Staining Kit (Product Code: G1018) Purchased from Wuhan Guge Biological Co., Ltd. (Hubei, China). BCA protein quantification kit was purchased from Beijing Soleibo Technology Co., Ltd. CD8a antibody (Product Code: PC0020). Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) antibodies (Product Code: ab13504 and ab53358), Toll-like receptor-4 antibody (Product Code: ab8376), AP-1 antibody (Product Code: ab21981) and NF-κB antibody (Product Code: ab16502), NF-AT antibody (Product Code: ab59204) IL-4 antibody (Product Code: ab9622) Purchased from Abcam (Cambridge, MA, USA). HP-1100 High Performance Liquid Chromatography (HPLC) was purchased from Agilent Technologies (Palo Alto, CA, USA). Rat estrogen receptor beta (ERβ) (Product Code: JL45045) and Rat estrogen receptor alpha (ERα) enzyme-linked immunosorbent assay kit was purchased from China Jianglai Biotechnology Co., Ltd (Shanghai, China). (Product Code: JL26212). Reverse transcriptase kit and amplification reagent kit were purchased from Takara (Bio, Inc., Japan, Product Code: 9109 and RR820A). Trizol reagent was purchased from Thermo-Invitrogen, USA (Product Code: 15596018). All other chemicals were commercially available.
This experiment was proved the Animal Experiment Ethics Committee of Zunyi Medical University [No. Lun Shen (2018) No. 2-166]. All experiments were conducted under the guidelines and regulations of Zunyi Medical University. To avoid the interference of internal estrogen, 60 male Sprague–Dawley rats of 4 weeks were provided by Hunan Changsha Tianqin Biological Technology Co., Ltd. [License No. SCXK (Xiang) 2014-0011].
After 1 week of adaptive feeding, they were randomly divided into 5 groups (control, 3 NP dose and E2 groups) with 12 animals in each group. The control group (C) was given corn oil 5 ml/kg daily by gavage, and the NP low (L), medium (M) and high (H) dose groups were given NP 0.4, 4, 40 mg/kg/day. The gavage volume was 5 ml/kg/bw/day and gavage was conducted at 8 o'clock every morning, and continued for 180 days. Animal room conditions were: room temperature: 25 ± 3 °C, 55 ± 5% humidity, 12 h dark/light cycle, free feeding and drinking. Since the plastic contains NP, the rats were fed in polyacrylamide cages to reduce interference.
Subchronic exposure dose setting
In the Pearl River of China, maximum NP content and sediment in water were 3 µg/l and 12 mg/kg, while sediment in fish was 9 mg/kg . The previous research of our lab showed that the 200 mg/kg of NP exposure affected the normal development of the immune organs of the rats’ offspring . In addition, The Danish Institute of Toxicology, Safety and Toxicology proposed the allowable daily intake of NP for humans was 5 μg/kg bw/day, and the tolerable daily intake of rats was calculated as 0.5 mg/kg bw according to the safety factor (human:rat = 1:100)/day. Since the tolerable daily NP intake in humans is 5 μg/kg bw/day, human exposure to NP amounts to 40 mg/kg/d were based on the maximum daily NP tolerance (5 μg bw/day) for an adult weighing 60 kg (this dose considers NP accumulation and the body’s long-term multi-pathway exposure to NP) [60, 67,68,69,70]. In this study, the human NP exposure dose was much lower than the 40 mg/kg/d NP exposure dose, and the tolerable daily intake of mice was calculated to be 0.5 mg/kg bw/day according to the safety factor (human:mice = 1:100). The other dose groups were 10 and 100 times higher than this dose group; hence, the NP doses were 0.4, 4, and 40 mg/kg bw/day, respectively. As reported, the 17α-ethinylestradiol concentrations in wastewater ranged from 0.00 to 0.52 ng/l, and the NP concentrations ranged from 287 to 2058 ng/l. The NP/17α-ethinylestradiol levels in wastewater were approximately 0–3958 times higher [71, 72]. The E2 treatment group dosage was set at 30 µg/kg regarding the maximum exposure concentration of NP and 17α-ethinylestradiol.
Coefficient of the rats body weight and the spleen
The weight of the rats was monitored before each gavage. After 180 days of overnight fasting, the rats were anesthetized with 20% urethane and their whole blood was collected from the abdominal aorta. After standing for 2 h, the blood serum was collected by centrifugation. The spleen tissues were quickly taken on ice, dried on filter papers and weighed. The pieces of 0.5 × 0.5 cm spleen was fixed in 10% formaldehyde. The remaining spleen tissues were frozen at − 80 °C for later use. The organ coefficient was calculated according to the formula C = (m1/m2) × 100%, where C represented the organ coefficient, m1 represented the wet weight of the spleen, and m2 represented the weight of the rat.
Pathological alterations of spleen tissue
After soaked in 10% formaldehyde for 24 h, the rat spleen tissues were dehydrated, and embedded in paraffin. The sections were stained with hematoxylin and eosin and observed with an optical microscope (HT7700, Hitachi, Tokyo, Japan). There were 3 replicates of each sample. Image-pro plus 6.0 software (Media Cybernetics, Rockville, MD) was used for quantitative analysis .
Approximately 1 cubic millimeter of rat spleen tissues were fixed in 2.5% neutral glutaraldehyde. The tissues were rinsed with 0.1 M phosphate buffered solution (PBS), dehydrated with an ascending graded alcohol series (30%, 50%, 70%, 90%, 100%), and then followed by permeation, embedding, curing, sectioning, electron staining. After drying, the sections were observed by a transmission electron microscope (Hitachi Co., Ltd., Tokyo, Japan) .
Detection of the immune inflammatory cells by Giemsa staining
One drop of fresh blood was added into the clean glass slide near the ground glass end, mixed with Jimsa A and Jimsa B, respectively, stained for about 3–5 min, rinsed the solution, and dried by air, and then about 200 cells were counted under the microscope .
Detection of serum ER-α, ER-β and IL-4 by enzyme-linked immunosorbent assay (ELISA)
Serum samples were prepared by centrifuging the whole blood samples. The serum was centrifuged after being stored in a refrigerator at 4 °C overnight. Serum IL-4, ER-α, ER-β levels were measured by ELISA kits under the manufacture’s protocols. Data were collected and calculated .
Detection of the serum levels of immunoglobulins (IgE, IgG and IgM)
One ml of the standard protein solution at the concentration 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mg/ml were added to 6 colorimetric tubes, respectively, with 3 parallel tubes for each concentration. 4 ml of Biuret reagent were added after making up to 3 ml of each tube. The measurement was carried out at the wavelength of 546 nm in the UV spectrometer (Unico UV-2100, Shanghai).
Detection of the typing of lymphocyte subsets by flow cytometric
The peripheral blood lymphocytes were suspended in 1 ml pre-cooled Stain Buffer (The composition of aqueous buffered solution was phosphate buffered solution (PBS) fetal bovine serum and ≤ 0.09% sodium azide) centrifuged at 300 g/min for 10 min at 4 °C. This step was repeated twice. Finally, the final cell concentration was adjusted to 2 × 107 cells/ml with pre-cooled Stain Buffer. 100 μl of the cell suspension was added to a flow tube, blotted with specific surface antibody, and incubated for 20 min in the dark (on ice). The cells were washed twice with Stain Buffer, 1 ml/tube/time, centrifuged at 300×g for 5 min. Finally the cells were resuspended with 0.5 ml Stain Buffer and tested on a flow cytometer (BD FACS Calibur, USA) [75, 76].
Detection of NP concentration in spleen by high-performance liquid chromatography (HPLC)
According to Yu et al. methods , 0.05 g spleen tissues were homogenized with 2 ml of n-hexane-diethyl ether (volume ratio 7:3), and centrifuged at 4000 rpm for 10 min. The supernatant was transferred to another clean glass tube, dried in a 40 °C water bath, and dissolved in 0.5 ml acetonitrile. The specimen, filtered with a 0.22 μm membrane (pre-rinsed with acetonitrile) into the sample bottle, was tested by a HP-1200 system (Agilent, SantaClara, CA, USA).
Detection of the expression levels of ER-α and ER-β proteins in spleen by immunohistochemical analysis
Paraffin sections were blocked with serum, blotted with primary antibody and secondary antibody. The nucleus were stained with DAPI. Finally, specimens were observed under a microscope and calculated Integral-Optical Density (IOD) [59, 77, 78].
Detection of the expression levels of AP-1, NF-AT and NF-KB proteins in spleen by immunofluorescence analysis
Paraffin sections were blocked with serum first, then marked by a histochemical pen and autofluorescence quencher was added to the sections followed by primary antibody and secondary antibody. The nuclei were stained with DAPI. Finally, specimens were observed under a fluorescence microscope and calculated Integral-Optical Density (IOD) [66, 77].
Detection of the expression levels of inflammatory-related genes (ER-α, ER-β, AP-1, NF-AT, NF-κB, IL-10 and IL-12) in spleen
50–100 mg of spleen tissues were homogenized with 1 ml of TRIzol (Invitrogen, USA) on ice and kept at room temperature for 10 min; 200 μl chloroform was added and centrifuged. RNA integrity was measured by mixing 1–20 µl of Qubit RNA IQ Assay kit samples RNA IQ working solution and then measuring on a Qubit 4 fluorometer. The results were expressed as RNA IQ values ranging from 1 to 10, similar to other RNA quality scores, with values closer to 10 indicating higher sample integrity. GAPDH was as the housekeeping gene, and the primer sequences are shown in Additional file 4: Table S1. The relative quantitative formula: Q = 2−ΔΔCt, −ΔΔCt = (average Ct value of target gene in experimental group-average Ct value of reference gene in experimental group) − (average Ct value of target gene in control group-average Ct value of reference gene in control group) was used to calculate the expression of each gene .
Detection of the expression levels of inflammatory-related proteins (ER-α, ER-β, AP-1, NF-AT, NF-κB, IL-10 and IL-12) in spleen
The expressions of synapse-associated proteins (ER-α, ER-β, AP-1, NF-AT, NF-κB, IL-10 and IL-12) in spleen tissues were detected by Western Blot. Total protein was extracted from 50 to 100 mg of spleen with 600 μl lysis buffer. Protein was analyzed by Image-lab (BIO-RAD, Version: 5.1.0) and Image software (JNIH, Bethesda, USA). GAPDH was as an internal reference to calculate the expression [71, 79].
Analysis of spleen tissue by RNA-Seq
The total RNA was extracted and RNA library was constructed using KAPA Stranded RNA-Seq Library Prep Kit (Illumina). mRNA-seq was conducted under the Illumina HiSeq X Ten sequencing platform, and the sequencing volume was set to 6G reads [80,81,82], and the results of RNA-Seq are shown in Additional file 5: Table S2.
All experimental data were shown as mean ± SEM. Statistical analysis was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Variance test and one-way analysis of variance (One-Way ANOVA) were used in two-group comparisons. For further pairwise comparisons between groups, if the variances were equal, the Tukey test was used for pairwise comparisons, and if the variances were not uniform, Dunnett’s T3 was used for pairwise comparisons. Pearson correlation analysis was used for the correlation of normal measurement data, and Spearman correlation analysis was used for the correlation of non-normal measurement data. P < 0.05 was considered statistically significant.
Effects of NP on body weight and organ coefficient in rats
There was an increased tendency of their body weight when rats exposed to NP for 30–120 days, but there was no significant difference in groups (P > 0.05). When rats were exposed to NP for 160 days, there was a significant difference in body weights in groups (F = 8.487, P < 0.001). However, compared with the control group, the weight of the spleen after 180 days of subchronic exposure to NP showed a tendency to decrease, and there were significant differences in groups (F = 12.166, P < 0.001), as shown in Fig. 1A, B.
Histopathologic alterations of spleen
The HE staining results of spleen tissues were analyzed. The number of lymphocytes and macrophages in the NP-exposed group was significantly reduced, the marginal zone of the follicle was obviously widened, plasma cell infiltration can be seen, and the central artery wall had a tendency to gradually thicken. The splenic sinus was congested and dilated, and a few neutrophil infiltrations were seen in the red pulp. Image-pro plus 6.0 software was used to quantitatively analyze the relative area of splenic white pulp, the relative area of splenic lymph nodes, and the number of spleen nodules in the spleen tissues of each group (Additional file 1: Fig. S1; Additional file 2: Fig. S2). The results showed the relative area of the spleen tissue of the rats in each NP group decreased compared with that of the control group (F = 26.03, P < 0.001). The relative area of lymph nodes in the M (NP, 4 mg/kg) and L (NP, 0.4 mg/kg) groups gradually decreased (F = 25.76, P < 0.001), and the number of spleen nodules decreased to a certain extent compared with the control group (F = 16.065, P < 0.001), as shown in Fig. 1C.
Effects of NP on the ultrastructure of spleen
In the control group lymphocyte nucleoplasm ratio was high, cytoplasm had a few organelles, mitochondria and rough endoplasmic reticulum were more and no obvious abnormalities. The lymphocytes in the Low NP group were slightly rich in cytoplasm, mitochondria were slightly swollen, part of the cristae was reduced and vacuolated, and the rough endoplasmic reticulum had no obvious abnormalities. In the Middle NP group, the lymphocyte nuclear chromatin agglomerated with agglomerated edges, cytoplasmic mitochondria were swollen, cristae were reduced, vacuolization, mitochondrial membrane was incomplete, and the rough endoplasmic reticulum was slightly expanded. In the High NP group, lymphocytes were slightly rich in cytoplasm, mitochondria were slightly swollen, some cristae were slightly reduced, focal vacuolation, and rough endoplasmic reticulum were slightly expanded. The nuclear chromatin in the estradiol group appeared clumping and pyknosis, as shown in Fig. 1D.
Effects of NP on the serum immune inflammatory cells
The number of neutrophils in the M and L NP groups increased compared to the control, and there were significant differences in the groups group (F = 21.35, P < 0.001). There were no significant difference of white blood cell count (F = 2.477, P = 0.077), lymphocyte count (F = 1.953, P = 0.141), monocyte count (F = 1.106, P = 0.369), and red blood cell count (F = 0.141, P = 0.965) among the groups. The results of cell counting are shown in Fig. 2A.
Comparisons of serum levels of immunoglobulins (IgE, IgG and IgM)
With the increase of the NP exposure dose, the serum levels of immunoglobulin IgG (F = 48.582, P < 0.001) and IgM (F = 5.709, P = 0.002) decreased relative to the control group, and the difference in the groups was statistically significant. There was an increasing trend of the serum immunoglobulin IgE content compared with the control group, and the differences among the groups were statistically significant (F = 93.559, P < 0.001), as shown in Fig. 2B.
Effects of NP on the typing of lymphocyte subsets
The percentage of Th cells (CD3+CD4+) in each NP-exposed group was significantly lower than that of the control group, and the difference in the groups was statistically significant (F = 27.137, P < 0.001). The percentage of Ts cells (CD3+CD8+) in each NP-exposed dose group was lower than the control group C group, and the difference among the groups was not statistically significant (F = 0.878, P = 0.491). Compared with the control group, the ratio of CD4+ to CD8+ in H NP group had a downward trend, and the difference was significant (F = 3.941, P = 0.013). The percentage of B lymphocytes (CD3−CD161+) in each NP-exposed group was significantly higher than that of the control group, and the difference between the groups was statistically significant (F = 39.814, P < 0.001). The percentage of NK cells (CD3−CD45+) in each NP-exposed group was lower than that in the control group, and the difference was not statistically significant (F = 83.746, P < 0.001), as shown in Fig. 2C, D.
Effects of NP on the serum levels of ER-α, ER-β and IL-4
The levels of IL-4 (F = 568.4, P < 0.001), ER-α (F = 38.9, P < 0.001), and ER-β (F = 31.51, P < 0.001) in serum were higher than those in the control group, and the differences among the groups were significant, as shown in Fig. 3A.
Effects of NP on the expression levels of ER-α and ER-β proteins in spleen
The ERα was mainly expressed in the nucleus of granulocytes (shown by the arrow), which was diffusely distributed and strongly positive. Compared with the control group, the ER-α staining in each NP group gradually increased with the increase of the NP dose (F = 189.3, P < 0.001). The ER-β was mainly expressed in the cytoplasm of splenic lymphocytes (shown by the arrow), which was widely distributed and strongly positive. As the NP dose increased, the intensity of staining of the ER-β gradually increased (F = 414.9, P < 0.001), as shown in Fig. 3B, C.
Effects of NP on the expression levels of AP-1, NF-AT and NF-KB proteins in spleen
The expressions of the three transcription factors positively expressed in the cytoplasm of splenic lymphocytes. Compared with the control group, the fluorescence staining intensity of NF-AT (F = 189.3, P < 0.001) and AP-1 (F = 354.9, P < 0.001) in the spleen of rats gradually increased in each NP group. The fluorescence intensity of NF-κB staining gradually weakened. The difference among the groups was significant (F = 413.8, P < 0.001), as shown in Fig. 3D, E.
Effects of NP on the expression levels of inflammatory-related genes (ER-α, ER-β, AP-1, NF-AT, NF-Κb, IL-10 and IL-12) in spleen
The mRNA expressions levels of ER-α (F = 10.95, P < 0.001), ER-β (F = 4.891, P = 0.005), IL-10 (F = 4.223, P = 0.01), AP-1 (F = 3.543, P = 0.02), NF-AT (F = 2.839, P < 0.05), IL-4 (F = 4.212, P = 0.01), TLR4 (F = 5.884, P = 0.002), NF-κB (F = 6.653, P < 0.001), IL-12 (F = 3.137, P = 0.02) and GATA-3 (F = 4.223, P = 0.01) in each NP group were more higher than in the blank control group (P < 0.05), as shown in Fig. 4A.
Effects of NP on the expression levels of inflammatory-related proteins (ER-α, ER-β, AP-1, NF-AT, NF-κB, IL-10 and IL-12) in spleen
Compared with the control group, the expression of ER-α (F = 193.8, P < 0.001), ER-β (F = 158.6, P < 0.001), NF-AT (F = 7.149, P < 0.001), IL-4 (F = 11.43, P < 0.001) and AP-1 (F = 27.94, P < 0.001) protein in the spleen gradually increased in each NP-exposed group, and there were statistical differences among the groups. The protein expressions of TLR4 (F = 90.81, P < 0.001), NF-κB (F = 162.5, P < 0.001) and IL-12 (F = 17.55, P < 0.001) gradually decreased, and the differences among the groups were statistically significant, as shown in Fig. 4B, C.
Analysis of spleen tissue by RNA-Seq
A total of 6 samples (3 biological replicates) of the control group and the NP exposure group were analyzed. The differentially expressed genes were selected by DESeq2. In this analysis, the screening conditions were P value < 0.05 and fold-change greater than 2 times and less than 0.5 times. The pathway analysis of the differential genes was based on the KEGG database (http://www.genome.jp/kegg/), and the pathways that were significantly related to the differential genes were screened from the database. In the final comparison, 810 differential transcripts were obtained, of which 363 genes were up-regulated and 447 genes were down-regulated. As shown in the volcano map (Fig. 5D), a correlation analysis heat map (Fig. 5A), and a cluster map of differential conversion rates (Fig. 5C) based on the differentially expressed genes, there was a significant difference between the control group and the NP group.
As shown in Fig. 5, the biological processes, cell composition, and molecular functions analyzed from GO were integrated through multiple functional levels of cells, tissues, organs, and species, combined with the significant difference P value, and pathway analysis (as shown in Fig. 5B, E) differential gene signal pathway analysis bubble diagram and gene signal pathway diagram (Fig. 5G, H), select significant cytokines, as shown in Fig. 5F, which were up-regulated: IL1R, TNF-a, IL10, TLR2, INOS, IL1β, CD18, CXCL4, down-regulated: TLR4, TLR3, TLR11, CD80, Gata-3, IL12, TL9R, CD21, TGF-α, IAP, XIAP. According to the specific log2FoldChange, P value, Padj value (as shown in Fig. 5F and Additional file 6: Table S3), the 3 up-regulated TNF-a, IL10, INOS, and the 3 down-regulated: TLR4, Gata-3, IL12 were finally screened.
NP concentration in the spleen of rat
When rats were subchronically exposed for 180 days, the content of NP of rats in the M and H groups has increased compared to the control group, and there were significant differences in the groups (F = 997.4, P < 0.001), as shown in Additional file 3: Fig. S3A, B.
Correlation analysis of NP concentration in rat spleen and relative expression of estrogen receptor, IL-4, NF-κB, AP-1 and other immune factors
The relative protein expressions of AP-1 (r = 0.74, P < 0.001), IL-4 (r = 0.508, P = 0.011), ER-β (r = 0.861, P < 0.001) and ER-α (r = 0.848, P < 0.001) were positively correlated with the concentration of NP in the spleen of rats exposed to NP for 180 days. The relative protein expressions of NF-κB (r = − 0.929, P = 0.015), IL-12 (r = − 0.804, P < 0.001) and TLR-4 (r = − 0.925, P < 0.001) were negatively correlated with the concentration of NP in the spleen of rats exposed to NP for 180 days, as shown in scatter plot (Fig. 4D).
As far as we know, this was the first time to explore the effects of low-dose NP exposure, combined with the regulation of estrogen receptors, NF-AT, AP-1, and NF-κB transcription factors, on the immune and inflammatory damage of the body. In this study, for the first time, transcriptome sequencing technology, was used to screen differential gene expression between the control group and the 40 mg/kg NP exposure group—in the spleen, using advanced methods, such as KEGG database and pathway analysis. The difference in RNA expression further revealed the specific effect of estrogen receptor and NP on the regulation of immune inflammation.
The Endocrine Society and the US Environmental Protection Agency (USEPA) proposed that EDCs have complicated influences on the immune system. Indeed, it is easy to overlook the no observed adverse effect level (NOAEL) [60, 81, 83, 84]. It is currently challenging to determine dangerous levels of NP exposure that have daily and long-term effects on humans due to influences by both exposure dose and exposure period (early exposure and longer term exposure) [85,86,87,88]. Immunotoxicology is a marginal discipline based on toxicology and immunology [84, 89]. Female mice are more susceptible to autoimmune diseases than male mice despite the pathological differences [28, 32]. In previous studies on the effects of NP on immune function, exposure levels far exceeded the actual environmental exposure levels, exposure periods were too short, and the selected species were primarily females. To accurately approximate the environmental exposure dose of NP in the general population, a concentration slightly higher than the ambient concentration (but in the same order of magnitude) was adopted [90,91,92,93]. Specifically, the doses were set at 0, 4, and 40 μg/kg day, and the duration of exposure was set at 180 d. SD male mice of stable species were selected to eliminate gender interference and explore lower doses and longer exposure. In addition, combining immune function alterations with gene expression changes. To explore the value of data support for gene expression profiles to predict changes in immune factors.
As the body’s largest immune organ, the spleen accounts for the largest amount of lymphatic tissue in the body, and is considered the body’s immune center [94,95,96]. All immunocytes accumulate, mainly producing immune responses to various antigenic substances and secreting many immune effectors. Indeed, organ weight positively correlated with cellular immune function [64, 97,98,99]. The pathological changes suggested that the subchronic exposure of NP caused atrophy and pathological irreversible damage to the spleen of rats, which could affect immune damage and abnormalities in regulatory functions. Although changes in body weight and organ coefficients were non-functional tests, their changes may be the earliest and most sensitive way to reflect the toxic effects of exogenous compounds [96, 100,101,102]. In this study, the changes in the organ coefficients of the NP groups were different from those of the control group. However, there were no significant differences in body weight during the first 160 days of intragastric administration, and the differences were significant after 160 days. The reason may be the lower exposure dose and the exposure time, indicating that the low toxic dose needed to be used for a longer time to better show the toxic effect.
The results of the lymphocyte typing in this study indicated that the subchronic exposure of NP has broken the immune balance of T, B, and NK cells in SD male rats. The ratio of CD4+/CD8+ reflected the state of immune balance, and its value decreased, which suggested that the initiation of immune response and the induction effect were also reduced [83, 86]. Some scholars have shown that NP can inhibit the mitosis of lymphocytes by acting on ER-α on mouse T cells and B cells, reducing the number of effector cells produced by T cells and B cells, and ultimately affecting the immune function [94, 103]. In addition, studies have found that estrogen stimulated expressions of IFN-γ and IL-12 in CD8+ T cells, thereby enhancing the activity of B lymphocytes [104, 105]. IL-12 is an important initiator of cellular immune responses, as demonstrated by high-throughput sequencing results detecting upregulation of the immune factors TNF-a and IL10 in IL12 downregulation experiments [106, 107]. Sundstedt has demonstrated that AP-1 and NF-κB transcription factors disturbed the expression of activated CD4+ T cells in vivo . The expression of AP-1, NF-κB, NF-AT, and octamer-binding transcription factors, is involved in the regulation of IL-2 gene promoter activity, resulting in the reduction of IL-2 expression at the mRNA and protein levels . Dokter et al. found that IL-10 and IL-4 inhibited the effect of LPS-induced IL-10 and IL-4 on activator protein-1 (AP-I), nuclear factor IL-6 (NF-IL6), and nuclear factor NF-κB expression by suppressing the transcriptional rate of the IL-6 gene . Alteration of these transcription factors might inhibit IL-2 expression in T cells. In addition, CD4+ T is the precursor of IL-2 and IL-4, and IL-2 could produce IL-4 [107, 110, 111].
According to the data of estrogen receptors ER-α and ER-β in immunohistochemical experiments, it could be inferred that subchronic low-dose exposure of NP has caused alterations in the expression of estrogen receptors, the balance and homeostasis of each lymphocyte population, and Th1/Th2 cell balance and CD4+/CD8+ ratio. Therefore, estrogen receptors may be involved in this process of disturbing the balance.
Peripheral blood is the main source of lymphocytes and can provide interspecies comparisons [55, 112]. The results of peripheral blood lymphocyte typing and immunoglobulin detection showed that white cells and monocytes in the peripheral blood of NP-exposed rats increased significantly, and the number of lymphocyte decreased. Serum IgM and IgG levels declined with the increase of NP exposure dose and serum IgE levels increased. All the above indicated that NP exposure had an immunosuppressive effect similar to E2, by reducing the body's ability to recognize, killing and promptly removing mutant cells from the body to prevent tumors and other diseases [113, 114]. Studies have shown that NP can inhibit mouse type 1 helper T cells (Thl) from secreting INF-γ and T cells (Th2) to produce IL-4, showing an immunosuppressive effect [103, 112]. The increase of IL-4 was related to the increase of IgE concentration in animal plasma [113,114,115,116]. This was consistent with the results of the Elisa experiment, immunoglobulin detection and peripheral blood test in this study.
When naive CD4+ T cells differentiate into effector Th2 cells, they are prone to allergic sensitization. IL-4 was secreted during the degranulation of eosinophils. In the Th2 differentiation stage, IL-4 played an important role [117, 118], which was the same as the results of increase of IL-4 in the spleen in immunofluorescence, RT-qPCR and western blot in this study. The normal expression of inflammatory factors is essential for body homeostasis as they may cause cellular or tissue damage and may induce major diseases (e.g., cancer and metabolic diseases) [54, 119,120,121]. As a key gene of the TLR family, TLR4 activates MAPK, PI3K, and NF-κB pathways, thus affecting cell activity and apoptosis [61, 122,123,124,125]. Relevant studies have shown that vitamin D reduced the inflammatory response and lung cell apoptosis in asthmatic mice through the high mobility group protein B1 (HMGB1)/TLR4/NF-kB) signaling pathway [124, 126]. Toll-like receptors (TLR2, 4) could interact with HMGB1 and the receptors of advanced glycation end products (RAGE) to activate the nuclear factor NF-κB signaling pathway and induce the release of downstream inflammatory mediators [59, 61, 126]. This also proved once again the pathway in this study was consistent to the asthma pathway and the TLR4-NF-κB pathway.
Immune factors can regulate many biological processes of hematopoietic cells and mediate cellular activation, differentiation and survival [34, 97, 98]. More and more studies show that, Involved in the induction of nitric oxide, the production of reactive oxygen species, etc. It is regulated by the mobilization of reactive calcium through the opening of Ca2+ channels on the intracellular calcium storage membrane by immune factor signals [29, 127]. GRP75-induced ER-mitochondrial Ca2+ transfer may be an important factor in Th1/Th2 imbalance in asthma patients. In addition, HMGB1 specifically promotes Th2 cytokines release through GRP75-induced enhancement of ER-mitochondrial Ca2+ transfer and increase in ROS . Oxidative stress, inflammation, and hypoxia can lead to proteins unfolding or misfolding that interfere with endoplasmic reticulum (ER) homeostasis, thereby triggering ER stress. ER stress and autophagy are mechanistically interrelated, and exposure to low doses of NP may cause stress and autophagy. The role of NP exposure in the tumor microenvironment regulation during immune damage has not been elucidated [129,130,131,132,133,134,135,136,137,138,139,140]. It has been demonstrated that the activation of C kinase 1 scaffold protein Receptor (RACK1), an EDC target in the immune milieu, is an important molecular player in cancer progression [141,142,143,144]. Urriola et al. reported that BPA and NP induce apoptosis in prostate and ovarian cancer cell lines, a process dependent on ADAM17 activation [145, 146]. Sun et al. found that epidermal growth factor receptor (EGFR) and ERK up-regulation is associated with estrogenic responses during 17α-ethinylestradiol- and 4-NP-stimulated mock lung adenocarcinoma cell production . Since microenvironmental dynamics highly influence tumor development, estrogen-sensitive peripheral cells, such as breast cancer cells and vascular endothelial cells, are also involved in angiogenesis and tumorigenesis [145, 146, 148,149,150,151]. Moreover, the tumor microenvironment itself contains stromal fibroblasts, endothelial cells, immunocytes, and acellular components of the extracellular matrix [152,153,154]. Hence, the specific mechanisms by which subchronic NP exposure causes chronic immune inflammation in mice, induces estrogen receptor and transcription factor changes in vivo, and disrupts the Th1/Th2 balance, CD4+/CD8+ ratio, and tumor microenvironmental homeostasis are complex and need to be further explored [145, 146].
The immune system is closely related to the nerve and endocrine systems, and the three systems influence each other to form a neuroendocrine immune network [51, 54, 64, 75, 99, 100, 155,156,157]. As an important sex steroid hormone, estrogen plays an important role in the occurrence and development of autoimmune diseases by directly regulating the development of immune organs, immune cells and the secretion of immune-related factors [11, 41, 56, 119]. However, the mechanism of estrogen receptor in NP on immune cytokines needs further research. In addition, technical NP contains over 20 para-substituted isomers. The estrogenic activity of the isomers is heavily dependent on facets of the structure of the nonyl side chain, such as the degree of branching and bulkiness. Only total level of 4-NP was detected rather than all the isomers in animal experiment, which is a limitation of our study [4,5,6,7].
Subchronic exposure of 40 mg/kg/day NP for 180 days can cause immune dysfunction in rats, resulting in immunotoxicity and inflammatory effects, and lead to changes in the activity of transcription factors, such as AP-1, NF-AT, NF-κB and differential immune inflammatory factors in rats. In this process, estrogen receptors may play an important role and participate in immune inflammatory by affecting the expressions of transcription factors and immune factors.
Availability of data and materials
The data sets used and/or analyzed during the current study available from the corresponding author on reasonable request.
Fu X, Xu J, Zhang R et al (2020) The association between environmental endocrine synthetic endocrine disrupting chemicals on the development and functions of human immune cells. Environ Int 125:350–364
Mao Z, Zheng XF, Zhang YQ et al (2012) Occurrence and biodegradation of nonylphenol in the environment. Int J Mol Sci 13(1):491–505
Lu Z, Gan J (2014) Analysis, toxicity, occurrence and biodegradation of nonylphenol isomers: a review. Environ Int 73:334–345
Klaus G, Volkmar H, Bjoern T et al (2002) Endocrine disrupting nonylphenols are ubiquitous in food. Environ Sci Technol 36(8):1676–1680
Klaus G, Einhard K, Bjoern T (2006) Estrogen-active nonylphenols from an isomer-specific viewpoint: a systematic numbering system and future trends. Anal Bioanal Chem 384(2):542–546
Torsten R, Bjoern T, Boehme Roswitha M et al (2011) Endocrine disrupting nonyl- and octylphenol in infant food in Germany: considerable daily intake of nonylphenol for babies. Chemosphere 82(11):1533–1540
Klaus G, Torsten R, Roswitha B (2017) An isomer-specific approach to endocrine-disrupting nonylphenol in infant food. J Agric Food Chem 65(6):1247–1254
Acir IH, Guenther K (2018) Endocrine-disrupting metabolites of alkylphenol ethoxylates—a critical review of analytical methods, environmental occurrences, toxicity, and regulation. Sci Total Environ 635:1530–1546
Huang SL, Tuan NN, Lee K (2016) Occurrence, human intake and biodegradation of estrogen-like nonylphenols and octylphenols. Curr Drug Metab 17(3):293–302
Soares A, Guieysse B, Jefferson B et al (2008) Nonylphenol in the environment: a critical reviewon occurrence, fate, toxicity and treatment in wastewaters. Environ Int 34(7):1033–1049
Rosenfeld CS, Cooke PS (2019) Endocrine disruption through membrane estrogen receptors and novel pathways leading to rapid toxicological and epigenetic effects. J Steroid Biochem Mol Biol 187:106–117
Bronowicka-Kłys DE, Lianeri M, Jagodziński PP (2016) The role and impact of estrogens and xenoestrogen on the development of cervical cancer. Biomed Pharmacother 84:1945–1953
Heldring N, Pike A, Andersson S et al (2007) Estrogen receptors: how do they signal and what are their targets. Physiol Rev 87(3):905–931
Labrie F, Labrie C, Bélanger A et al (2001) Pure selective estrogen receptor modulators, new molecules having absolute cell specificity ranging from pure antiestrogenic to complete estrogen-like activities. Adv Protein Chem 56:293–368
Kuiper GG, Lemmen JG, Carlsson B et al (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139(10):4252–4263
Barrett JR (2014) EDCs and estrogen receptor activity: a pathway to safer chemical design. Environ Health Perspect 122(12):A339
Edwards M, Dai R, Ahmed SA et al (2018) Our environment shapes us: the importance of environment and sex differences in regulation of autoantibody production. Front Immunol 9:478
Castles CG, Oesterreich S, Hansen R et al (1997) Auto-regulation of the estrogen receptor promoter. J Steroid Biochem Mol Biol 62(2–3):155–163
Taylor SE, Martin-Hirsch PL, Martin FL (2010) Oestrogen receptor splice variants in the pathogenesis of disease. Cancer Lett 288(2):133–148
Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F (2014) Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids 90:13–29
Grimaldi C, Cleary J, Dagtas AS et al (2002) Estrogen alters thresholds for B cell apoptosis and activation. J Clin Investig 109(12):1625–1633
Escribese MM, Kraus T, Rhee E et al (2008) Estrogen inhibits dendritic cell maturation to RNA viruses. Blood 112(12):4574–4584
Kitawaki J, Kado N, Ishihara H et al (2002) Endometriosis: the pathophysiology as an estrogen-dependent disease. J Steroid Biochem Mol Biol 83(1–5):149–155
Merrheim J, Villegas J, Van Wassenhove J et al (2020) Estrogen, estrogen-like molecules and autoimmune diseases. Autoimmun Rev 19(3):102468
Malayer JR, Cheng J, Woods VM (1999) Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1. Biol Reprod 60(5):1204–1210
Confavreux CB, Fontana A, Guastalla JP et al (2007) Estrogen-dependent increase in bone turnover and bone loss in postmenopausal women with breast cancer treated with anastrozole. Prevention with bisphosphonates. Bone 41(3):346–352
Pollard KM (2012) Gender differences in autoimmunity associated with exposure to environmental factors. J Autoimmun 38(2–3):J177–J186
Ahmed SA, Hissong BD, Verthelyi D, Donner K, Becker K, Karpuzoglu-Sahin E (1999) Gender and risk of autoimmune diseases: possible role of estrogenic compounds. Environ Health Perspect 107(Suppl 5):681–686
Molero L, García-Durán M, Diaz-Recasens J et al (2002) Expression of estrogen receptor subtypes and neuronal nitric oxide synthase in neutrophils from women and men: regulation by estrogen. Cardiovasc Res 56(1):43–51
Gear RB, Belcher SM (2017) Impacts of bisphenol a and ethinyl estradiol on male and female CD-1 mouse spleen. Sci Rep 7(1):856
Pabbidi MR, Kuppusamy M, Didion SP, Sanapureddy P, Reed JT, Sontakke SP (2018) Sex differences in the vascular function and related mechanisms: role of 17β-estradiol. Am J Physiol Heart Circ Physiol 315(6):H1499–H1518
Ye RR, Peterson DR, Shin-Ichi K et al (2018) Sex-specific immunomodulatory action of the environmentalestrogen 17α-ethynylestradiol alongside with reproductive impairment in fish. Aquat Toxicol 203:95–106
Yoshino S, Yamaki K, Li X et al (2004) Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice. Immunology 112(3):489–495
Wang YX, Gu ZW, Cao ZW et al (2019) Nonylphenol can aggravate allergic rhinitis in a murine model by regulating important Th cell subtypes and their associated cytokines. Int Immunopharmacol 70:260–267
Kazuma Y, Shuji O, Yonako N et al (2003) Effects of various chemicals including endocrine discuptors and analogs on the secretion of Th1 and Th2 cytokines from anti CD3-stimulated mouse spleen cells. J Health Sci 49(3):195–204
Stossi F, Singh PK, Mistry RM et al (2022) Quality control for single cell imaging analytics using endocrine disruptor-induced changes in estrogen receptor expression. Environ Health Perspect 130(2):27008
Pellegrini M, Bulzomi P, Lecis M et al (2014) Endocrine disruptors differently influence estrogen receptor β and androgen receptor in male and female rat VSMC. J Cell Physiol 229(8):1061–1068
Shanle EK, Xu W (2011) Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action. Chem Res Toxicol 24(1):6–19
Lakshmanan MD, Shaheer K (2020) Endocrine disrupting chemicals may deregulate DNA repair through estrogen receptor mediated seizing of CBP/p300 acetylase. J Endocrinol Invest 43(9):1189–1196
Park MA, Hwang KA, Lee HR et al (2011) Cell growth of BG-1 ovarian cancer cells was promoted by 4-tert-octylphenol and 4-nonylphenol via downregulation of TGF-β receptor 2 and upregulation of c-myc. Toxicol Res 27(4):253–259
Kim YS, Hwang KA, Hyun SH et al (2015) Bisphenol A and nonylphenol have the potential to stimulate the migration of ovarian cancer cells by inducing epithelial–mesenchymal transition via an estrogen receptor dependent pathway. Chem Res Toxicol 28(4):662–671
Rastgar S, Movahedinia A, Salamat N et al (2018) Interruption of immune responses in primary macrophages exposed to nonylphenol provides insights into the role of ER and NF-KB in immunotoxicity of persian sturgeon. Fish Shellfish Immunol 86:125–134
Francis DA, Karras JG, Ke XY, Sen R, Rothstein TL (1995) Induction of the transcription factors NF-kappa B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int Immunol 7(2):151–161
Hedin KE, Bell MP, Kalli KR, Huntoon CJ, Mckean DJ (1997) Delta-opioid receptors expressed by jurkat t cells enhance il-2 secretion by increasing ap-1 complexes and activity of the nf-at/ap-1-binding promoter element. J Immunol 159(11):5431
Pei XC, Li G, Zhang YM et al (2012) The role of activated T cell nuclear factor in the regulation of IL-4 expression by DEHP. Chin Public Health 028(011):1449–1452
Park J, Chung SW, Kim SH et al (2006) Up-regulation of interleukin-4 production via NF-AT/AP-1 activation in T cells by biochanin A, a phytoestrogen and its metabolites. Toxicol Appl Pharmacol 212(3):188–199
Lacour M, Arrighi JF, Müller KM, Carlberg C, Saurat JH, Hauser C (1994) cAMP up-regulates IL-4 and IL-5 production from activated CD4+ T cells while decreasing IL-2 release and NF-AT induction. Int Immunol 6(9):1333–1343
Mukherjee U, Samanta A, Biswas S, Ghosh S, Das S, Banerjee S, Maitra S (2022) Chronic exposure to nonylphenol induces oxidative stress and liver damage in male zebrafish (Danio rerio): mechanistic insight into cellular energy sensors, lipid accumulation and immune modulation. Chem Biol Interact 351:109762
Lee MH, Park J, Chung SW et al (2004) Enhancement of interleukin-4 production in activated CD4+ T cells by diphthalate plasticizers via increased NF-AT binding activity. Int Arch Allergy Immunol 134(3):213–222
Rooney JW, Hoey T, Glimcher LH (1995) Coordinate and cooperative roles for NF-AT and AP-1 in the regulation of the murine IL-4 gene. Immunity 2(5):473–483
Kuo CH, Yang SN, Kuo PL et al (2012) Immunomodulatory effects of environmental endocrine disrupting chemicals. Kaohsiung J Med Sci 28(7 Suppl):S37–S42
Bahadar H, Abdollahi M, Maqbool F et al (2015) Mechanistic overview of immune modulatory effects of environmental toxicants. Inflamm Allergy Drug Targets 13(6):382–386
Milla S, Depiereux S, Kestemont P (2011) The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review. Ecotoxicology 20(2):305–319
Nowak K, Jabłońska E, Ratajczak-Wrona W et al (2019) Immunomodulatory effects of synthetic endocrine disrupting chemicals on the development and functions of human immune cells. Environ Int 125:350–364
Phiel KL, Henderson RA, Adelman SJ et al (2005) Differential estrogen receptor gene expression in human peripheral blood mononuclear cell populations. Immunol Lett 97(1):107–113
McLachlan JA (2016) (2008) Environmental signaling: from environmental estrogens to endocrine-disrupting chemicals and beyond. Andrology 4(4):684–694
Lélu K, Laffont S, Delpy L et al (2011) Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis. J Immunol 187(5):2386–2393
Feng F, Ma H, Yao Y, Wang C, Zhang L, Cheng L et al (2016) Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol a treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells. Oncotargets Ther 9:3451
Wang J, Li R, Peng Z, Hu B, Rao X, Li J (2020) HMGB1 participates in LPS-induced acute lung injury by activating the AIM2 inflammasome in macrophages and inducing polarization of M1 macrophages via TLR2, TLR4, and RAGE/NF-κB signaling pathways. Int J Mol Med 45(1):61–80
Nielsen E, Stergaard G, Thorup I, Ladefoged O, Jelnes JE (2000) Toxicological evaluation and limit values for nonylphenol, nonylphenol ethoxylates, tricresyl, phosphates and benzoic acid
Gu W, Wang Y, Qiu Z, Dong J, Wang Y, Chen J (2018) Maternal exposure to nonylphenol during pregnancy and lactation induces microglial cell activation and pro-inflammatory cytokine production in offspring hippocampus. Sci Total Environ 634:525–533
Bennasroune A, Rojas L, Foucaud L, Goulaouic S, Laval-Gilly P, Fickova M et al (2012) Effects of 4-nonylphenol and/or diisononylphthalate on thp-1 cells: impact of endocrine disruptors on human immune system parameters. Int J Immunopathol Pharmacol 25(2):365–376
Aune TM, Flavell RA (1997) Differential expression of transcription directed by a discrete NF-AT binding element from the IL-4 promoter in naive and effector CD4 T cells. J Immunol 159(1):36–43
Wang P, Wang J, Sun YJ, Yang L, Wu YJ (2017) Cadmium and chlorpyrifos inhibit cellular immune response in spleen of rats. Environ Toxicol 32(7):1927–1936
Fan JJ, Wang S, Tang JP et al (2019) Bioaccumulation of endocrine disrupting compounds in fish with different feeding habits along the largest subtropical river, China. Environ Pollut 247(APR):999–1008
Xu W, Yu J, Li S et al (2020) Depressive behavior induced by nonylphenol and its effect on the expression of ER-α and ER-β in nerve cells of rats. J Affect Disord 263:373–381
Li S, Xu W, Gong L et al (2021) Subchronic nonylphenol exposure induced anxiety-like behavior and decreased expressions of regulators of synaptic plasticity in rats. Chemosphere 282:130994
Ying GG, Kookana RS, Kumar A, Mortimer M (2009) Occurrence and implications of estrogens and xenoestrogens in sewage effluents and receiving waters from South East Queensland. Sci Total Environ 407(18):5147–5155
Mes TD, Zeeman G, Lettinga G (2005) Occurrence and fate of estrone, 17β-estradiol and 17α-ethynylestradiol in stps for domestic wastewater. Rev Environ Sci Bio/Technol 4(4):275–311
Ying GG, Williams B, Kookana R (2002) Environmental fate of alkylphenols and alkylphenol ethoxylates—a review. Environ Int 28(3):215–226
Yu J et al (2020) Effect of gestational and lactational nonylphenol exposure on airway inflammation in ovalbumin-induced asthmatic rat pups. Chemosphere 250:126244
Kwack SJ, Kwon O, Kim HS, Kim SS, Kim SH, Sohn KH, Lee RD, Park CH, Jeung EB, An BS, Park KL (2002) Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo. J Toxicol Environ Health A 65(5–6):419–431
Wang J, Li T, Cai H et al (2021) Protective effects of total flavonoids from Qu Zhi Qiao (fruit of Citrus paradisi cv. Changshanhuyou) on OVA-induced allergic airway inflammation and remodeling through MAPKs and Smad2/3 signaling pathway. Biomed Pharmacother 138:111421
Yu J et al (2019) Indoor PM from coal combustion aggravates ovalbumin-induced asthma-like airway inflammation in BALB/c mice. Am J Physiol Lung Cell Mol Physiol 317:29–38
Hargreaves CE, Salatino S, Sasson SC et al (2021) Decreased ATM function causes delayed DNA repair and apoptosis in common variable immunodeficiency disorders. J Clin Immunol 41(6):1315–1330
Lyons AB, Parish CR (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171(1):131–137
Yamaguchi H, Hiroi M, Mori K et al (2021) Simultaneous expression of Th1 and Treg-associated chemokine genes and CD4, CD8, and Foxp3 cells in the premalignant lesions of 4NQO-induced mouse tongue tumorigenesis. Cancers 13(8):1835
Zhao Y, Fu B, Chen P et al (2021) Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti-Thy-1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway. Cell Prolif 54(6):e13055
Yang S, Shi X, Li X et al (2019) Oriented collagen fiber membranes formed through counter-rotating extrusion and their application in tendon regeneration. Biomaterials 207:61–75
Li Y, Cheng M, Zhao Y et al (2021) Effects of fluoride on PIWI-interacting RNA expression profiling in testis of mice. Chemosphere 269:128727
Guido S, Laura S, Franziska B et al (2004) Discriminating different classes of toxicants by transcript profiling. Environ Health Perspect 112(12):1236–1248
Luebke RW, Holsapple MP, Ladics GS et al (2006) Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process. Toxicol Sci 94(1):22–27
Descotes J (2004) Importance of immunotoxicity in safety assessment: a medical toxicologist’s perspective. Toxicol Lett 149(1–3):103–108
Myers JP, Zoeller RT, Vom Saal FS (2009) A clash of old and new scientific concepts in toxicity, with important implications for public health. Environ Health Perspect 117(11):1652–1655
Cunny HC, Mayes BA, Rosica KA, Trutter JA, Van Miller JP (1997) Subchronic toxicity (90-day) study with para-nonylphenol in rats. Regul Toxicol Pharmacol 26(2):172–178
Paurene D, Holland NT (2011) Biomarkers of immunotoxicity for environmental and public health research. Int J Environ Res Public Health 8(5):1388–1401
Brennan FR, Morton LD, Spindeldreher S et al (2010) Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies. MAbs 2(3):233–255
Heindel JJ, Skalla LA, Joubert BR, Dilworth CH, Gray KA (2017) Review of developmental origins of health and disease publications in environmental epidemiology. Reprod Toxicol 68:34–48
House RV (1999) Theory and practice of cytokine assessment in immunotoxicology. Methods 19(1):17–27
Xia H (2012) Study on the effect of nonylphenol on the immune system function of female SD rats. Soochow University
Udoji F, Martin T, Etherton R, Whalen MM (2010) Immunosuppressive effects of triclosan, nonylphenol, and DDT on human natural killer cells in vitro. J Immunotoxicol 7(3):205–212
Quanxiang M, Xuehui F, Zeshan M (2004) The effect of nonylphenol on the immune function of mice. Chin Public Health 02:77–78
Yao G, Hu Y, Liang J et al (2005) Nonylphenol-induced thymocyte apoptosis is related to Fas/FasL pathway. Life Sci 77(26):3306–3320
Sakazaki H, Ueno H, Nakamuro K (2002) Estrogen receptor alpha in mouse splenic lymphocytes: possible involvement in immunity. Toxicol Lett 133:221–229
Pei X, Duan Z, Ma M et al (2014) Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes. Mol Biol Rep 41(4):2129–2142
Coffey G, DeGuzman F, Inagaki M, Pak Y, Delaney SM, Ives D, Betz A, Jia ZJ, Pandey A, Baker D, Hollenbach SJ, Phillips DR, Sinha U (2012) Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis. J Pharmacol Exp Ther 340(2):350–359
Ward S, Marelli-Berg F (2009) Mechanisms of chemokine and antigen-dependent t-lymphocyte navigation. Biochem J 418(1):13–27
Nieto M, Frade J, Sancho D et al (1997) Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis. J Exp Med 186(1):153–158
Bansal A, Henao-Mejia J, Simmons RA (2018) Immune system: an emerging player in mediating effects of endocrine disruptors on metabolic health. Endocrinology 159(1):32–45
Yang Y, Meng K, Chen M et al (2021) Fluorotelomer alcohols’ toxicology correlates with oxidative stress and metabolism. Rev Environ Contam Toxicol 256:71–101
Ren R, Sun DJ, Yan H, Wu YP, Zhang Y (2013) Oral exposure to the herbicide simazine induces mouse spleen immunotoxicity and immune cell apoptosis. Toxicol Pathol 41(1):63–72
Vos JG, Loveren HV (1994) Developments of immunotoxicology methods in the rat and applications to the study of environmental pollutants. Toxicol In Vitro 8(5):951–956
Farrar JD, Asnagli H, Murphy KM (2002) T helper subset development: roles of instruction, selection, and transcription. J Clin Investig 109(4):431–435
Berg RE, Cordes CJ, Forman J (2015) Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18. Eur J Immunol 32(10):2807–2816
Mehrotra PT, Wu D, Crim JA et al (1993) Effects of IL-12 on the generation of cytotoxic activity in human CD8+ T lymphocytes. J Immunol 151(5):2444–2452
Green VL, Irune E, Prasai A, Alhamarneh O, Greenman J, Stafford ND (2012) Serum IL10, IL12 and circulating CD4+CD25high T regulatory cells in relation to long-term clinical outcome in head and neck squamous cell carcinoma patients. Int J Oncol 40(3):833–839
Larmonier N, Marron M, Zeng Y, Cantrell J, Romanoski A, Sepassi M, Thompson S, Chen X, Andreansky S, Katsanis E (2007) Tumor-derived CD4(+)CD25(+) regulatory T cell suppression of dendritic cell function involves TGF-beta and IL-10. Cancer Immunol Immunother 56(1):48–59
Sundstedt A, Sigvardsson M, Leanderson T, Hedlund G, Kalland T, Dohlsten M (1996) In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors. Proc Natl Acad Sci USA 93(3):979–984
Dokter WH, Koopmans SB, Vellenga E (1996) Effects of IL-10 and IL-4 on LPS-induced transcription factors (AP-1, NF-IL6 and NF-kappa B) which are involved in IL-6 regulation. Leukemia 10(8):1308–1316
Luo CY, Wang L, Sun C, Li DJ (2011) Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro. Cell Mol Immunol 8(1):50–58
Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA (2007) IL-2 is essential for TGF-beta to convert naive CD4+CD25- cells to CD25+Foxp3+ regulatory T cells and for expansion of these cells. J Immunol 178(4):2018–2027
Santa K, Ohsawa T, Sakimoto T (2016) Para-nonylphenol induces apoptosis of u937 human monocyte leukemia cells in vitro. Endocr Metab Immune Disord Drug Targets 16(3):213–223
Okunuki H, Teshima R, Sakushima J et al (2000) Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice. Immunol Lett 74(3):233–237
Choi IH, Shin YM, Park JS et al (1998) Immunoglobulin E-dependent active fatal anaphylaxis in mast cell-deficient mice. J Exp Med 188(9):1587–1592
Kabesch M, Tzotcheva I, Carr D, Höfler C, Weiland SK, Fritzsch C, von Mutius E, Martinez FD (2003) A complete screening of the IL4 gene: novel polymorphisms and their association with asthma and IgE in childhood. J Allergy Clin Immunol 112(5):893–898
Kabesch M, Schedel M, Carr D, Woitsch B, Fritzsch C, Weiland SK, von Mutius E (2006) IL-4/IL-13 pathway genetics strongly influence serum IgE levels and childhood asthma. J Allergy Clin Immunol 117(2):269–274
Busse W, Corren J, Lanier BQ et al (2001) Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J Allergy Clin Immunol 108(2):184–190
Schroeder JT, MacGlashan DW Jr, Kagey-Sobotka A et al (1994) IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures. J Immunol 153(4):1808–1817
Lee JW, Han HK, Park S et al (2017) Nonylphenol increases tumor formation and growth by suppressing gender-independent lymphocyte proliferation and macrophage activation. Environ Toxicol 32(6):1679–1687
Medzhitov R (2008) Review article origin and physiological roles of inflammation. Nature 7203:428–435
Gu W, Wang Y, Qiu Z, Dong J et al (2018) Maternal exposure to nonylphenol during pregnancy and lactation induces microglial cell activation and pro-inflammatory cytokine production in offspring hippocampus. Sci Total Environ 634(Spec):525–533
Cipelli R, Harries L, Okuda K et al (2014) Bisphenol A modulates the metabolic regulator oestrogen-related receptor-α in T-cells. Reproduction 147(4):419–426
Iwata M, Eshima Y, Kagechika H et al (2004) The endocrine disruptors nonylphenol and octylphenol exert direct effects on T cells to suppress Th1 development and enhance Th2 development. Immunol Lett 94(1–2):135–139
Zhang H, Yang N, Wang T et al (2018) Vitamin D reduces inflammatory response in asthmatic mice through HMGB1/TLR4/NF-κB signaling pathway. Mol Med Rep 17(2):2915–2920
Sar A, Stockhammer OW, Laan C et al (2006) Myd88 innate immune function in a zebrafish embryo infection model. Infect Immun 74(4):2436
Sun X, Zeng H, Wang Q, Yu Q, Wu J, Feng Y, Deng P, Zhang H (2018) Glycyrrhizin ameliorates inflammatory pain by inhibiting microglial activation-mediated inflammatory response via blockage of the HMGB1-TLR4-NF-kB pathway. Exp Cell Res 369(1):112–119
Swindle EJ, Metcalfe DD (2007) The role of reactive oxygen species and nitric oxide in mast cell-dependent inflammatory processes. Immunol Rev 217(1):186–205
Lv Y, Li Y, Zhang D, Zhang A, Guo W, Zhu S (2018) HMGB1-induced asthmatic airway inflammation through GRP75-mediated enhancement of ER-mitochondrial Ca2+ transfer and ROS increased. J Cell Biochem 119(5):4205–4215
Lin Z, Ru S (2018) Research advances on the molecular mechanism of environmental endocrine disrupting chemicals on promoting breast cell proliferation. Res Environ Sci 31(5):796–804
Smith M, Wilkinson S (2017) ER homeostasis and autophagy. Essays Biochem 61(6):625–635
Hu P, Han Z, Couvillon AD, Kaufman RJ, Exton JH (2006) Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol 26(8):3071–3084
Wilkinson S (2019) ER-phagy: shaping up and destressing the endoplasmic reticulum. FEBS J 286(14):2645–2663
Stephani M, Picchianti L, Dagdas Y (2021) C53 is a cross-kingdom conserved reticulophagy receptor that bridges the gap betweenselective autophagy and ribosome stalling at the endoplasmic reticulum. Autophagy 17(2):586–587
Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol 10:173–194
Qi Z, Chen L (2019) Endoplasmic reticulum stress and autophagy. Adv Exp Med Biol 1206:167–177
So JS (2018) Roles of endoplasmic reticulum stress in immune responses. Mol Cells 41(8):705–716
Spencer BG, Finnie JW (2020) The role of endoplasmic reticulum stress in cell survival and death. J Comp Pathol 181:86–91
Cao SS, Luo KL, Shi L (2016) Endoplasmic reticulum stress interacts with inflammation in human diseases. J Cell Physiol 231(2):288–294
Zhao H, Dong F, Li Y, Ren X, Xia Z, Wang Y, Ma W (2021) Inhibiting ATG5 mediated autophagy to regulate endoplasmic reticulum stress and CD4+ T lymphocyte differentiation: mechanisms of acupuncture’s effects on asthma. Biomed Pharmacother 142:112045
Xie M, Liang JL, Huang HD, Wang MJ, Zhang T, Yang XF (2019) Low doses of nonylphenol promote growth of colon cancer cells through activation of ERK1/2 via G protein-coupled receptor 30. Cancer Res Treat 51(4):1620–1631
Buoso E, Galasso M, Ronfani M, Papale A, Galbiati V, Eberini I, Marinovich M, Racchi M, Corsini E (2017) The scaffold protein RACK1 is a target of endocrine disrupting chemicals (EDCs) with important implication in immunity. Toxicol Appl Pharmacol 325:37–47
Buoso E, Galasso M, Serafini MM, Ronfani M, Lanni C, Corsini E, Racchi M (2017) Transcriptional regulation of RACK1 and modulation of its expression: role of steroid hormones and significance in health and aging. Cell Signal 35:264–271
Buoso E, Masi M, Galbiati V, Maddalon A, Iulini M, Kenda M, Sollner Dolenc M, Marinovich M, Racchi M, Corsini E (2020) Effect of estrogen-active compounds on the expression of RACK1 and immunological implications. Arch Toxicol 94(6):2081–2095
Racchi M, Buoso E, Ronfani M, Serafini MM, Galasso M, Lanni C, Corsini E (2017) Role of hormones in the regulation of RACK1 expression as a signaling checkpoint in immunosenescence. Int J Mol Sci 18(7):1453
Urriola-Muñoz P, Lagos-Cabré R, Patiño-García D, Reyes JG, Moreno RD (2018) Bisphenol-A and nonylphenol induce apoptosis in reproductive tract cancer cell lines by the activation of ADAM17. Int J Mol Sci 19(8):2238
Noorimotlagh Z, Mirzaee SA, Martinez SS, Rachoń D, Hoseinzadeh M, Jaafarzadeh N (2020) Environmental exposure to nonylphenol and cancer progression risk—a systematic review. Environ Res 184:109263
Sun CH, Chou JC, Chao KP, Chang HC, Lieu FK, Wang PS (2019) 17α-Ethynylestradiol and 4-nonylphenol stimulate lung adenocarcinoma cell production in xenoestrogenic way. Chemosphere 218:793–798
Arneth B (2019) Tumor microenvironment. Medicina (Kaunas) 56(1):15
Baghban R, Roshangar L, Jahanban-Esfahlan R, Seidi K, Ebrahimi-Kalan A, Jaymand M, Kolahian S, Javaheri T, Zare P (2020) Tumor microenvironment complexity and therapeutic implications at a glance. Cell Commun Signal 18(1):59
Balkwill FR, Capasso M, Hagemann T (2012) The tumor microenvironment at a glance. J Cell Sci 125(Pt 23):559
Burks H, Pashos N, Martin E, Mclachlan J, Bunnell B, Burow M (2017) Endocrine disruptors and the tumor microenvironment: a new paradigm in breast cancer biology. Mol Cell Endocrinol 457:13–19
Wu Q, Zhou X, Li P et al (2021) ROC1 promotes the malignant progression of bladder cancer by regulating p-IκBα/NF-κB signaling. J Exp Clin Cancer Res 40(1):158
Scsukova S, Rollerova E, Bujnakova MA (2016) Impact of endocrine disrupting chemicals on onset and development of female reproductive disorders and hormone-related cancer. Reprod Biol 16(4):243–254
Masi M, Racchi M, Travelli C, Corsini E, Buoso E (2021) Molecular characterization of membrane steroid receptors in hormone-sensitive cancers. Cells 10(11):2
Straub RH, Cutolo M, Buttgereit F, Pongratz G (2010) Energy regulation and neuroendocrine-immune control in chronic inflammatory diseases. J Intern Med 267(6):543–560
Cravedi JP, Zalko D, Savouret JF, Menuet A, Jégou B (2007) Le concept de perturbation endocrinienne et la santé humaine [The concept of endocrine disruption and human health]. Med Sci 23(2):198–204 (in French)
Giulivo M, Lopez de Alda M, Capri E, Barceló D (2016) Human exposure to endocrine disrupting compounds: their role in reproductive systems, metabolic syndrome and breast cancer. A review. Environ Res 151:251–264
This work was supported by the National Natural Science Foundation of China (22166035); Guizhou High-Level Innovative Talent Support Program (2022, 6014); the Scientific and Technological Talent Support Program of the Educational Commission of Guizhou Province of China (KY054); 15851 Project Talent in Zunyi municipal government, Guizhou Province (2018(E-262)); Science and Technology Foundation of the Health Commission of Guizhou Province (gzwkj2021-39, gzwkj2021-536).
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The Ethics Committee of the Zunyi Medical University approved the study (2015-1-017). All methods were performed in accordance with guidelines and regulations of the Zunyi Medical University.
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All the authors read and approved this paper.
The authors declare that they have no competing interests.
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Additional file 1: Figure S1.
4200 instrument result graph.
Additional file 2: Figure S2.
Peak diagram of sample quality test results.
Additional file 3: Figure S3.
Detecting the accumulation of NP in the spleen by HPLC (n = 6).
Additional file 4: Table S1.
Additional file 5: Table S2.
Quality analysis of spleen tissue samples by RNA-Seq.
Additional file 6: Table S3.
Reference value of immune cytokines.
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Fu, X., Xu, J., Ni, C. et al. Effects of subchronic exposure of nonylphenol on the expression of immune-related factors and estrogen receptors in the spleen of rats. Environ Sci Eur 34, 30 (2022). https://doi.org/10.1186/s12302-022-00610-6
- Immune-related factors
- Estrogen receptor