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. 2021 Aug 28;184(1):142–153. doi: 10.1093/toxsci/kfab105

The Impact of Di-Isononyl Phthalate Exposure on Specialized Epithelial Cells in the Colon

Karen Chiu 1,2, Shah Tauseef Bashir 3,4, Justin Chiu 2,4, Romana A Nowak 4,5, Jodi A Flaws 1,2,4,5,✉
PMCID: PMC8677456  PMID: 34453847

Abstract

Di-isononyl phthalate (DiNP) is a high-molecular-weight phthalate commonly used as a plasticizer for polyvinyl chloride and other end products, such as medical devices and construction materials. Most of our initial exposure to DiNP occurs by ingestion of DiNP-contaminated foods. However, little is known about the effects of DiNP on the colon. Therefore, the goal of this study was to test the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon. To test this hypothesis, adult female mice were orally dosed with corn-oil vehicle control or doses of DiNP ranging from 20 µg/kg/d to 200 mg/kg/d for 10–14 days. After the dosing period, mice were euthanized in diestrus, and colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a Paneth cell marker (Lyz1), and estradiol levels in sera compared with control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (eg, goblet and Paneth cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon.

Keywords: di-isononyl phthalate, colon, inflammation, endocrine disruption, specialized epithelial cells

High-molecular-weight phthalates are a large class of chemicals that have 7–13 carbons in the ester side chains (European Chemical Industry Council, 2018). Phthalates are used as plasticizers for polyvinyl chloride (PVC) and other end products such as medical devices (ie, catheters, tubing, and gloves), flooring, wall coverings, roofing, cable and wires, coated fabrics, and vinyl clothing. High-molecular-weight phthalates include di(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DiNP). Phthalates make up the largest segment of the plasticizer market—specifically two-thirds of the plasticizer market (BCC Research, 2020). DEHP was a popular high-molecular-weight phthalate, but it was marked as a concerning substance by the European Parliament and by Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) legislation under Regulation No. 1907/2006 in 2008. Specifically, REACH regulations in the European Union (EU) have restricted the use of DEHP in toys, consumer goods, and childcare articles and approved the use of DiNP for use in all applications. The United States Consumer Product Safety Commission has also restricted the use of several phthalates, including DEHP, di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP), in toys and childcare products (U.S. Consumer Product Safety Commission, 2019).

Evidence has shown that DEHP is endocrine-disrupting and carcinogenic in multiple organ systems and animals (Caldwell, 2012; Kavlock et al., 2006; Rowdhwal and Chen, 2018). Because of this, DEHP is commonly replaced with DiNP, which has very similar plasticizer properties to DEHP. DiNP is thought to be a useful substitute because it has a higher molecular weight than DiNP, and thus, it is less volatile than DEHP. The market size of DiNP is projected to increase from 2.7 billion USD in 2019 to 3.2 billion USD by 2024 at a compound annual growth rate of 4.0% (Markets and Markets, 2019). As the DiNP market size increases, its production and contamination in the environment are bound to be significant. In fact, DiNP and its metabolites have already been detected in human urine and sweat all around the world, which shows that humans are ubiquitously exposed to DiNP (Genuis et al., 2012; Saravanabhavan and Murray, 2012).

The primary route of DiNP exposure in humans is through oral ingestion of DiNP contaminated foods (National Research Council (US) Committee on the Health Risks of Phthalates, 2008). A study has shown that after oral administration, DiNP was quickly absorbed by the gastrointestinal (GI) tract in rats (McKee et al., 2002). Specifically, more than 90% of the DiNP was absorbed by the GI tract within 1 h (McKee et al., 2002). Further, 24 h after a final dose of DiNP, 16–23% of the administered DiNP dose remained in the GI tract (McKee et al., 2002). Most of the DiNP was metabolized into the monoester, mono-isononyl phthalate (MiNP), and was excreted primarily in urine and some in feces after 24 h (McKee et al., 2002).

Because most DiNP exposure initially targets the gut, the purpose of this study was to investigate the impact of DiNP exposure on the colonic immune microenvironment as well as the impact DiNP has on specialized epithelial cells. A few previous studies have investigated the impacts of DiNP on the intestines. One study showed that oral exposure to 380 mg/kg/day DiNP caused severe villous atrophy in the small intestines compared with control in gestating and lactating rats (Setti Ahmed et al., 2018). The other study showed that subacute DiNP exposure in adult female mice altered intestinal morphology at environmentally-relevant and toxicological doses compared with control (Chiu et al. 2020). The same study also showed that DiNP exposure significantly altered immune responses in the colon by decreasing cytokine soluble intercellular adhesion molecule-1 (sICAM-1) and increasing Tnf expression compared with control (Chiu et al., 2020). DiNP exposure also significantly decreased estradiol levels in the colon compared with control (Chiu et al., 2020).

Several intestinal epithelial cells play a role in the colonic immune microenvironment including goblet, Paneth, enteroendocrine, and absorptive epithelial cells. Specifically, goblet cells play a role in innate immune response by producing mucins that act as a barrier, trap pathogens, and secrete anti-microbial peptides, cytokines, and chemokines (Knoop and Newberry, 2018). Antibacterial peptides secreted from goblet cells include resistin-like molecular beta (RELM-β) and are responsible for killing microbes that penetrate the mucus layer (Morampudi et al., 2016; Propheter et al., 2017). Like goblet cells, Paneth cells play a big role in secreting antimicrobial peptides and immunomodulating proteins (Lueschow and McElroy, 2020). Enteroendocrine cells are typically known to detect nutrient status and mediate digestion by releasing peptide hormones. However, they are also key sensors of microbial metabolites and can release cytokines in response to pathogen-associated molecular patterns (PAMPs) (Worthington et al., 2018). Absorptive epithelial cells provide a barrier against pathogens and mediate innate immune responses. However, when innate immune responses fail, epithelial cells can help with early adaptive immune responses by presenting antigens to dendritic cells (Schleimer et al., 2007).

Many environmental chemicals have been shown to alter the gut microbe population (Chiu et al., 2020). A significant change in the gut microbe population can trigger specific toll-like receptors (TLRs) to activate the innate immune response by recognizing microbes and their microbial products. Mice have 12 different TLRs (TLR1–TLR9, TLR11–TLR13) (Kawasaki and Kawai, 2014). Out of the 12 members of mouse TLRs, only a few TLRs recognize microbial membrane components. For example, TLR4 recognizes lipopolysaccharide from the outer membrane of gram-negative bacteria, and TLR5 recognizes bacterial flagellin (Kawasaki and Kawai, 2014). TLR4 and TLR5 are both cell surface receptors and their activation leads to nuclear factor- κB (NF-κB) signaling (Gupta et al., 2014; Seki and Brenner, 2008). Activation of NF-κB ultimately leads to pro-inflammatory cytokine production that activates the innate immune system. Phthalate exposure has been shown to alter the gut microbiome in several organisms including humans, mice, rats, and zebrafish (Adamovsky et al., 2020; Hu et al., 2016; Lei et al., 2019; Yang et al., 2019); therefore, it is possible that DiNP-induced dysbiosis can impact the expression and activation of TLRs.

Estradiol plays a significant role in regulating immune responses. Generally, females have lower infection rates than males because they have stronger innate and adaptive inflammatory responses compared with males, which can result in faster clearance of pathogens (Klein and Flanagan, 2016; Ortona et al., 2016). However, women also have higher rates of autoimmune diseases compared with men (Ortona et al., 2016), likely due to the role of the dominant female hormone, estradiol, and the expression of estrogen receptors 1 and 2 (ESR1 and ESR2) in immune cells (Straub, 2007). Phthalates have been shown to significantly alter sex hormone levels compared with control (Barakat et al., 2017; Chiang et al., 2020), making it is plausible that estradiol plays a role in DiNP-induced immune responses in the colon. Thus, the current study investigated immune responses in the colon by examining the effects of DiNP on estradiol levels, gene expression or protein levels of specialized epithelial cell types, and gene expression or protein levels of several immune cells and their factors. Specifically, the study tested the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon.

MATERIALS AND METHODS

 

 

Chemicals

DiNP (Sigma-Aldrich, St. Louis, Missouri, CAS No. 28553-12-0) and tocopherol-stripped corn oil (MP Biomedicals, Solon, Ohio) were used in the dosing experiment. The DiNP doses were prepared as previously described (Chiu et al., 2020). Briefly, corn oil was used as vehicle control. Corn oil was also used to serially dilute DiNP into the following doses for the animals: 0.02, 0.2, 2, 20, and 200 mg/kg/day DiNP. The two lowest doses of DiNP (0.02 and 0.2 mg/kg) were selected to mimic environmentally-relevant exposures in humans (Bogen et al., 2001; Hines et al., 2012; Silano et al., 2019), whereas the higher doses of DiNP (2, 20, and 200 mg/kg) were selected to understand toxicological dose responses of DiNP (Chiang et al., 2020; Ma et al., 2014; Qin et al., 2020; Waterman et al., 2000) and mimic exposure levels found in foods (Kiralan et al., 2020; Silano et al., 2019).

Experimental animals

All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee (IACUC) and conducted in AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-approved facilities (Protocol No.: 20034 and 19110). Adult CD-1 mice (female, 2 months old) were purchased from Charles River (Wilmington, MA) and were allowed to acclimate to environmentally controlled facilities (21.1 ± 2.2 C, 12 h light: 12 h dark, 50 ± 20% humidity) for 7 days. During the acclimation and dosing period, mice were fed reverse-osmosis-treated water and 8604 Tekland Rodent Diet ad libitum.

Experimental design

Adult female mice (36 mice total; n = 6 mice per treatment group) were orally exposed to corn-oil vehicle or varying doses of DiNP (0.02, 0.2, 2, 20, and 200 mg/kg/day). This was done by gently pipetting the chemical into the mouth to mimic the primary exposure route of phthalates in humans. The mice were dosed every morning for a minimum of 10 days and then euthanized during diestrus by CO2 asphyxiation and cervical dislocation. Estrous cyclicity was determined by vaginal lavage using diluted phosphate-buffered saline. If mice were not in diestrus, the mice were continually dosed for a maximum of 14 days until they were in diestrus.

Tissue collection

Distal colons were collected, trimmed of mesenteric tissues, and flushed with cold PBS (1X) to remove colonic contents. Tissues were snap-frozen in liquid nitrogen and stored at −80°C for RNA and cytokine extraction.

Histology, immunohistochemistry, and image analysis

Separate sections of the distal colons were fixed in 10% formalin (Macron Fine Chemicals, Center Valley, Pennsylvania) for 24 h, and then the 10% formalin was replaced with 70% ethanol for at least 24 h. After 24 h, tissues were embedded in paraffin blocks. Colonic tissues were sectioned at 7 µm thickness using a Microm HM 310 and mounted onto glass slides for immunohistochemistry.

Immunohistochemistry was performed on slides sliced from paraffin-embedded blocks at 7 µm and dried at room temperature overnight. Slides were deparaffinized using xylene for 5 min (×3) followed by decreasing concentration of ethanol washes of 100% (×2), 90%, 80%, and 70% for 3 min each. Then, slides were rinsed in tap water and washed in 1X phosphate-buffer saline (PBS). Heat-induced epitope retrieval was conducted in a hot water bath (100°C) using citrate buffer pH 6.0 for 30 min. After antigen retrieval, slides were washed again with PBS. Next, endogenous peroxidases were blocked for 30 min using 0.3% hydrogen peroxide (H2O2). Tissues then were blocked with 10% normal goat serum (NGS) and 5% bovine serum albumin for 1 h and incubated with primary antibody overnight at 4°C in a humidity chamber. The primary rabbit polyclonal antibodies used in this study were Ki67 (Abcam ab15580, 1:500 dilution) and MUC2 (Abcam ab97386, 1: 500). On the next day, the slides were washed with PBS (×3) for 5 min and incubated with peroxidase labeled goat anti-rabbit secondary antibody (Vector Laboratories, PI-1000-1; 1:800) in PBS and 1% NGS for 1 h at room temperature and developed with 3,3’-diaminobenzidine (Vector Laboratories, SK4100). The slides were counterstained with hematoxylin. The counterstaining protocols with hematoxylin were optimized, and no differences in hematoxylin intensity were observed between slides.

Slides were scanned using NDP.scan 3.2.15 compatible with the Hamamatsu NanoZoomer 2.0 HT (Model #C9600-12). Images were viewed using NDP.view.2 software and then exported as TIFF images for semi-quantitative image analysis on Image J FIJI. Quantification of Ki67 was done by counting the number of positively stained cells in four full-length crypts from three sections per animal. Crypts that looked circular or damaged from tissue processing and sectioning were omitted. Values were reported as the number of positive Ki67 cells per crypt. Quantification of MUC2 was carried out according to Crowe and Yue (Crowe and Yue, 2019). In short, immunohistochemical images were deconvoluted using the plugin called “Colour Deconvolution” and a vector called “H DAB.” The ratio of brown (DAB) intensity to blue (hematoxylin) intensity was calculated and normalized to control. Furthermore, the data calculation for the brown color took area into consideration. Residual debris in the lumen of the colon was excluded from the image analysis through a cutting function on FIJI.

RNA extraction and gene expression analysis

RNAs from colon tissues were extracted using the RNeasy Micro Kit by Qiagen (Qiagen, Inc., Valencia, California). Instructions for RNA extraction were followed according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop (NanoDrop ND-1000, ThermoScientificTM, Waltham, Massachusetts). Samples were stored at −80°C until further use.

Complementary DNA (cDNA) synthesis was done by reverse transcribing 600 ng of RNA into DNA using iScript Reverse Transcriptase (Bio-Rad Laboratories, Inc., Hercules, California) on the Biometra T1 Thermocycler. Real-time quantitative polymerase chain reactions (RT-qPCR) were done in duplicate, so that each replicate contained 2 µl of cDNA, 5 pmol of forward and reverse primers for the gene of interest, SsoFast EvaGreen Supermix (Bio-Rad), and nuclease-free water (final volume of 10 µl). RT-qPCR were carried out on the CFX96 Real-Time Detection System (Bio-Rad Laboratories) and CFX Manager Software.

The qPCR primers used in this study are listed in supplementary materials (Table 1). Specifically, the following primers were used: toll-like receptor 4 (Tlr4), toll-like receptor 5 (Tlr5), mucin 1 (Muc1), mucin 2 (Muc2), mucin 3a (Muc3a), mucin 4 (Muc4), leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), chromogranin A (ChgA), lysozyme 1 (Lyz1), cluster of differentiation 24 (Cd24a), and villin 1 (Vil1). Expression of these genes of interest was normalized to corresponding values for β-Actin, a housekeeping gene. Relative fold changes of gene expression were calculated using the Pfaffl method (Pfaffl, 2001). Once the relative fold changes were calculated, they were normalized to the control group.

Hormone measurements

Blood was collected in a heparinized needle via cardiac puncture following euthanasia in the diestrous stage of the cycle. Blood samples were kept at room temperature for 15 min, so that samples could clot. After clotting, the blood samples were kept on ice until all samples for the day were collected. Sera were obtained from the blood samples by centrifugation at 14 000 RPM for 15 min at 4°C. Sera were stored at −20°C until further use. Estradiol sensitive enzyme-linked immunosorbent assay (ELISA) RUO kits were purchased from DRG (EIA-4399R), and ELISAs were carried out according to the manufacturer’s instructions. The assay dynamic range for estradiol was between 1.40 and 200 pg/ml. Samples below the limit of detection were analyzed using the lowest limit of detection value, which is 1.40, divided by the square root of two.

Cytokine extraction and measurements

Prior to cytokine extractions from the colon, tissues were weighed using an analytical scale. Then, tissues were resuspended in antifoam SE-15 (Millipore Sigma, St. Louis, Missouri) and tissue protein extraction reagent (ThermoFisher, Rockford, Illinois) and homogenized using the Tissue-Tearor (Model 985370-398) by Biospec Product, Inc. Following homogenization, the suspension was centrifuged for 10 min (6,000 rpm at 4°C). Protein concentrations were determined from the supernatant collected using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific).

Samples from each treatment group were pooled and subjected to the R&D Systems Proteome Profiler Cytokine Array to screen for cytokines that are detectable in colon tissues and to semi-quantitatively determine whether DiNP exposure altered cytokine levels in the colon compared with control. Statistical significance could not be determined because samples of the same treatment group were pooled together. Based on the results from the cytokine array, we determined whether observed changes between treatment groups were statistically different by conducting ELISAs using five to six independent samples per group. Because the cytokine array detected differences in CXCL12 and IL1-ra between treatment groups, we conducted CXCL12/SDF-1 (R&D systems, Minneapolis, Minnesota, MCX120) and IL-1RA ELISAs (R&D Systems, DY480) according to the manufacturers’ instructions. The ELISA assays were read with the MQX200 UQuant microplate reader (BioTek).

Statistical analysis

Data analyses were performed on Graphpad Prism 9.0.2, and the data were expressed as means ± standard error of the means (SEM). Outliers in the estradiol assay were identified and removed from analysis via ROUT. No outliers were identified in remaining data.

The data were analyzed using the Shapiro-Wilk test to assess for normality and lognormality. Data that were normally distributed and met the assumption for homogeneity of variance (HOV) were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s two-sided test. The following data were analyzed using this method: semi-quantification of MUC2 and Ki67 and expression of Muc1, Muc3a, Muc4, and Tlr4. Data that were not normally distributed and did not meet the assumption for HOV were analyzed using nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. The following data were analyzed using this method: Muc2 and estradiol levels. Statistical significance was assigned at p < .05 and is denoted with one or two asterisks. Borderline significance was assigned at 0.05 ≤ p < .10 and is denoted with a caret symbol.

RESULTS

 

Immunohistochemistry

A previous study in our laboratory showed that subacute exposure to DiNP ranging from 20 µg/kg to 20 mg/kg in adult female mice did not change the colonic gene expression of Ki67 compared with control (Chiu et al., 2020). To expand on the previous study, the current study investigated whether DiNP exposure altered the protein levels of Ki67 (a cell proliferation marker). Interestingly, DiNP exposure at all the tested doses significantly increased Ki67 protein, a marker of cell proliferation in the colons compared with control (Figure 1). The current study also examined immunostaining of MUC2 (a goblet cell marker) in the colon. DiNP exposure at 200 µg/kg to 200 mg/kg significantly increased immunostaining of MUC2 in the colon compared with control (p > .05, Figure 2).

Figure 1.

Figure 1.

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Protein levels of Ki67 in the colon. Immunohistological images (A) and quantification (B) of Ki67 were compared between control and di-isononyl phthalate (DiNP)-exposed colons. The data are presented as means ± standard error of the mean (SEM). Statistical significance is defined at p < .0001 and indicated with four asterisks (****). N = 6/treatment group.

Figure 2.

Figure 2.

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Protein levels of MUC2 in the colon. Immunohistological images (A) and quantification (B) of MUC2 were compared between control and di-isononyl phthalate (DiNP)-exposed colons. The data are presented as means ± standard error of the mean (SEM). Statistical significance is defined at p < .01 and indicated with two asterisks (**). N = 6/treatment group.

Gene Expression of Immune and Immune-Related Factors

The current study showed that DiNP exposure significantly increased the protein levels of Ki67 (a cell proliferation marker) compared with control and that most of the positive Ki67 cells were primarily observed at the bottom of the crypts (Figure 1). Thus, the next step was to investigate whether DiNP exposure altered gene expression in specialized epithelial cells in the crypts (ie, intestinal stem cells, Paneth cells, and other crypt cells) compared with control. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), lysozyme 1 (Lyz1), and cluster of differentiation 24 antigen (Cd24a) are all genes that code for epithelial cells found toward the bottom of intestinal crypts. LGR5 is a validated biomarker for identifying stem cells in the colon and small intestine (Gracz et al., 2013), LYZ1 is a marker for Paneth cells (Yu et al., 2018), and CD24a is an epithelial cell marker for cells located toward the bottom of intestinal crypts (Gracz et al., 2013). DiNP exposure did not affect the expression of Lgr5 and Cd24a in the colon compared with control (Figure 3). However, DiNP exposure at 200 mg/kg significantly increased the expression of Lyz1 compared with control (Figure 3).

Figure 3.

Figure 3.

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Gene expression of markers for various specialized epithelial cell types in the colon. The genes examined include Lgr5 (an intestinal stem cell marker), Vil1 (an intestinal cell-specific Ca2+-regulated-actin marker), Cd24a (an intestinal crypt cell marker), Lyz1 (a Paneth cell marker), and ChgA (an enteroendocrine cell marker). The data are presented as means ± standard error of the mean (SEM). Statistical significance is defined at p < .05 and indicated with an asterisk (*). N = 6/treatment group.

Gene expression of other specialized epithelial cells examined in this study were chromogranin A (ChgA) and villin 1 (Vil1). CHGA is a marker for mature enteroendocrine cells (Sei et al., 2011), and VIL1 is a marker for the epithelium border (Esmaeilniakooshkghazi et al., 2020). Similar to Lgr5 and Cd24a expression, DiNP exposure did not significantly alter ChgA and Vil1 expression compared with control (Figure 3).

Mucins are secreted from goblet cells to trap microbes and serve as a nutrient source to microbes. The mucins examined in this study included mucin 1, mucin 2, mucin 3a, and mucin 4. All of these mucins are found in the colon, but mucin 2 is the major mucin produced in the colon (Lillehoj et al., 2013). DiNP exposure did not significantly alter the expression of mucin 1, 2, 3a, and 4 in the colon compared with control (Figure 4).

Figure 4.

Figure 4.

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Relative fold differences of gastrointestinal mucins, Muc1, Muc2, Muc3a, and Muc4, in control and di-isononyl phthalate (DiNP)-treated colons. The data are presented as means ± standard error of the mean (SEM). N = 6/treatment group.

TLRs can recognize conserved areas derived from microbes, thereby initiating an innate immune response (Kawasaki and Kawai, 2014). Mice have 12 different types of TLRs (TLR1-9, 11-13). In this study, we specifically examined gene expression of Tlr4 and Tlr5 because these are some of the receptors that recognize microbes (Feuillet et al., 2006). DiNP exposure did not significantly alter expression of Tlr4 and Tlr5 in the colon compared with control (p > .05, Figure 5).

Figure 5.

Figure 5.

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Gene expression of Toll-like receptors, Tlr4 and Tlr5, in control and di-isononyl phthalate (DiNP)-exposed colons. Gene expression for each treatment group is compared with corn-oil vehicle control. The data are presented as means ± standard error of the mean (SEM). N = 6/treatment group.

Protein Levels of Immune and Immune-Regulating Factors

To determine the cytokine signature of DiNP-induced immune responses, a cytokine array was conducted to screen for the protein levels of 40 different cytokines (six samples from the same treatment group were pooled into one sample per treatment group). The cytokine array detected several cytokines, including IL-1RA and CXCL12 (Figure 6A). Through the cytokine array, we observed that DiNP exposure decreased protein levels of CXCL12 compared with control. Depending on the dose of DiNP, levels of IL-1RA in the colon increased or decreased compared with control (Figs. 6A and 6B). At environmentally relevant doses (20 and 200 µg/kg/day), DiNP exposure increased protein levels of IL-1RA in the colon compared with control. On the other hand, the top 3 highest doses of DiNP (2, 20, and 200 mg/kg) decreased protein levels of IL-1RA in the colon compared with control. Because samples were pooled for the cytokine array, we conducted IL-1RA and CXCL12 ELISAs to determine if differences between groups were statistically significant. Although differences were observed with the cytokine array on pooled samples, the ELISAs revealed that DiNP exposure did not statistically alter CXCL12 and IL-1RA levels in the colon compared with control (p > .05, Figs. 6C and 6D). Interestingly, the CXCL12 ELISA results follow a similar decreasing trend in response to DiNP as shown on the immunoblot on pooled samples.

Figure 6.

Figure 6.

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Protein levels of various cytokines. Protein levels of cytokines in the colons of adult female CD-1 mice exposed to corn oil control or di-isononyl phthalate (DiNP) (20 µg/kg/day—200 mg/kg/day) were first assessed by a cytokine array (A). The cytokine array was quantified using ImageJ (B). Values were pooled from six samples. The three pairs of prominent dots in the upper left, lower left, and lower right corner are positive controls. Following cytokine immunoblot screening, protein levels of cytokines were also measured by IL-1RA ELISAs (C) and CXCL12 ELISAs (D) in control and DiNP-exposed colons to determine whether the difference was statistically significant. The data are presented as means ± standard error of the mean (SEM). N = 4–6/treatment group.

Estradiol

A previous study from our laboratory showed that subacute exposure to DiNP significantly decreased estradiol levels in the distal colons of adult, female mice (Chiu et al., 2020). To determine whether the decrease in colonic estradiol was attributed to circulating estradiol levels, the current study measured estradiol levels in sera. DiNP exposure at 20 mg/kg significantly increased estradiol levels in sera compared with control (Figure 7). DiNP exposure at other doses (20 µg/kg, 200 µg/kg, 2 mg/kg, and 200 mg/kg) did not significantly alter circulating estradiol compared with control (Figure 7).

Figure 7.

Figure 7.

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Estradiol levels in the sera of adult female mice. Circulating levels of estradiol are reported as picograms of estradiol per milliliter (ml) of sera in control and treatment groups. The data are presented as means ± standard error of the mean (SEM). Statistical significance reported with two asterisks indicates that p < .01. N = 6/treatment group.

DISCUSSION

In this study, we showed that DiNP exposure significantly altered the colon microenvironment by increasing protein levels of a proliferation marker (Ki67) and expression of a goblet cell marker (MUC2) and a Paneth cell marker (Lyz1) (Figure 8). One previous study by Setti Ahmed et al. showed that subchronic exposure to DEHP and DEP increased Ki67 positive cells in the small intestine in gestational and lactating rats, but DiNP exposure did not affect the number of proliferating cells in the small intestine compared with control (Setti Ahmed et al., 2018). Although the study by Setti Ahmed et al. contradicts what was discovered in this study, it is important to note that proliferation in this study was examined in the colon rather than the small intestine. Further, Setti Ahmed et al. used DEHP and DEP, whereas the current study used DiNP. Several other studies indicate that DiNP exposure has significant proliferative effects in the uterus (Li et al., 2020), mouse splenocytes (Koike et al., 2010), and liver (Valles et al., 2003) compared with control. Furthermore, a previous study showed that subacute exposure in adult female mice did not significantly alter Ki67 expression compared with control (Chiu et al., 2020). The differences in genomic expression from a previous study and the proteomic expression from the current study could be attributed to the cell-level protein resolution of immunohistochemistry in the current study compared with the bulk measurement of gene expression by qPCR in the previous study. Although it is known that genomic and proteomic expression of Ki67 correlate in cell culture (Uxa et al., 2021), conditions change in vivo. Furthermore, genomic and proteomic studies have shown Ki67 gene expression and protein levels are poorly correlated (Koussounadis et al., 2015). Overall, phthalate exposure studies have shown that proliferation is significantly impacted by phthalate exposure compared with control; however, the effects vary depending on the model used, tissue examined, and the specific phthalate and dose used in the experiments.

Figure 8.

Figure 8.

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A graphic illustration depicting the impact of di-isononyl phthalate (DiNP) exposure on the colon. In a control or normal colon microenvironment, the colon shows basal levels of cytokines and chemokines and normal numbers of specialized epithelial cells. The control environment also shows goblet cells that secrete mucins and create a protective mucus layer. However, DiNP exposure alters the colonic microenvironment by increasing MUC2 mucin secretion and thus, creating a larger, thicker mucus barrier to potentially prevent further epithelial damage and infiltration of pathogens. DiNP exposure also significantly increases gene expression of Lyz1 compared with control, which could potentially increase the numbers of Paneth cells. Although the current study showed no changes in the cytokines examined, a previous study showed that DiNP altered gene expression and protein levels of various cytokines (Chiu et al., 2020), which may act on the intestinal epithelium and change cellular function and behavior, such as proliferation, apoptosis, and mucin production. In addition, DiNP exposure did not alter markers for stem and enteroendocrine cells. Collectively, the changes observed in the current study and previous studies (Chiu et al., 2020; Koike et al., 2010) could lead to increases in immune responses or serve as a protective mechanism to prevent further injury.

This study also showed that DiNP exposure (0.20, 2, 20, and 200 mg/kg) significantly increased MUC2 levels compared with control. The increase in MUC2 could be an indication of amplified gut mucosal protection through increased mucin production, creating a larger, thicker mucus barrier to potentially prevent further epithelial damage and infiltration of pathogens (Birchenough et al., 2015). Mucin 2 is the main mucin found in the colon and serves as a protective barrier against microbes. Less commonly known is that goblet cells also participate in innate immunity by secreting anti-microbial peptides, cytokines, and chemokines (Knoop and Newberry, 2018). Although the gene expression and protein levels of cytokines examined in this study were not statistically different between DiNP and control groups, it is possible that the cytokines entered the systemic circulation or that the increased production of mucins helped neutralize the DiNP-related effects and prevented further inflammation (Kim and Khan, 2013).

An interesting observation is that low doses of DiNP exposure induce maximum effects on Ki67 and MUC2 protein levels. Increasing the dose of DiNP does not create a more drastic effect on protein and gene expression in the colon compared with the lower doses. This unconventional dose-response relationship is also known as a non-monotonic dose-response relationship (NMDR) and is defined when the slope changes direction within the range of tested doses (Lagarde et al., 2015; Vandenberg, 2014; Vandenberg et al., 2012). NMDR curves are common patterns or relationships seen with endocrine-disrupting chemicals (Lagarde et al., 2015; Vandenberg, 2014; Vandenberg et al., 2012). In this study, it seems that DiNP has a binary response. In the case of MUC2 expression, 20 µg/kg DiNP does not have an effect on MUC2 in the colon. However, when the threshold (between 20 and 200 µg/kg DiNP) is met, all the higher doses (200 µg/kg to 200 mg/kg DiNP) have the same effect. In the case of Ki67 expression, DiNP exposures at all doses have the same effect. The binary response is not evident with Ki67 probably because threshold of response was already met.

The Paneth cell marker, Lyz1, was also significantly increased in the 200 mg DiNP treatment group compared with control. Paneth cells have an interesting epithelial lineage because they are the only type of cell that migrates downward (instead of upward toward the intestinal lumen) as they mature. Paneth cells contain eosinophilic granules in their cytoplasm, and thus, mediate inflammatory responses. In humans, Paneth cells are typically present in the small intestine and are absent in the colon. However, in disease conditions such as inflammatory bowel disease, Paneth cell numbers are significantly increased compared with healthy controls (Simmonds et al., 2014). Similar to humans, mice do not have Paneth cells in the colon under healthy conditions (Treuting et al., 2018). However, stressful conditions such as DiNP exposure increased the expression of Lyz1, suggesting a need for immune responses to neutralize the effects of DiNP. Furthermore, changes in the intestinal epithelium induced by DiNP exposure may impact how cytokines, chemokines, and other anti-microbial peptides are secreted and thus, impact DiNP-induced immune responses. Alternatively, cytokines and chemokines may act on the intestinal epithelium and change cellular function and behavior, such as proliferation, apoptosis, and mucin production of the specialized epithelial cells in the colon.

Studies have demonstrated that DiNP exposure disrupts estradiol levels locally and systemically (Chiu et al., 2020; Chiang et al., 2020). A previous study from our laboratory showed that subacute exposure to DiNP in adult female mice significantly decreased estradiol levels in the distal colon compared with control (Chiu et al., 2020). In a separate experiment from our laboratory, DiNP exposure (100 µg/kg and 200 mg/kg) also significantly decreased estradiol levels in sera compared with control (Chiang et al., 2020). Conversely, the current study showed that DiNP exposure significantly increased circulating estradiol levels compared with control. Possible explanations for these results are that estradiol synthesis by the gut affects circulating levels of estradiol or estradiol synthesis by the ovary affects levels found in circulation and the gut. However, the former explanation does not seem plausible because our current results showed that DiNP exposure increased circulating estradiol levels, and our previous study showed DiNP exposure decreased estradiol levels in the gut (Chiu et al., 2020). It is possible, however, that there is a compensatory decrease in the gut-synthesized estradiol when systemic levels of estradiol are high. The ovary is the major site of estradiol synthesis and therefore, contributes the most estradiol found in circulation (Barakat et al., 2016; Cui et al., 2013). Although estradiol synthesized by the gut can enter circulation, it is likely that DiNP exposure affects the ovary, thereby impacting estradiol synthesis (Chiang et al., 2020). Estradiol synthesized by the gut can enter circulation, which could further increase estradiol levels found in sera and further decrease estradiol levels found in the colon. A possible explanation for this occurrence is that estradiol is shuttled to other sites for the purposes of immune activation through estrogen signaling. The conflicting results on the effects of DiNP on circulating levels of estradiol may be explained by different experimental designs, and thus a different dosing regimen and total dose of DiNP used in the experiments. Furthermore, the ages of mice were different in these experiments compared with the previous study (Chiang et al., 2020). Because the ages of the female mice were different in these experiments, this adds another variable to the estradiol measurements because estradiol levels change with age (Frederiksen et al., 2020; Bell, 2018). The key takeaway is that DiNP exposure significantly disrupts estradiol levels systemically and locally, and the levels found in sera and colon may be independent of each other.

The results of our current study provide insights as to how much protection the gut provides against DiNP-induced effects and to what degree DiNP exposure impacts the colon. Our findings suggest that DiNP exposure increases the differentiation of goblet cells to increase the production of mucins, leading to increased immune responses, which may neutralize the effects of DiNP and prevent further injury to the gut (Figure 8). The increased expression of the Paneth cell marker, Lyz1, could potentially help with immune responses as well. The increase in estradiol levels may serve as another mechanism to increase immune responses as estrogens (including estradiol) have immunoenhancing effects (Klein and Flanagan, 2016). Most immune cells have estrogen receptors, and when estrogens bind to estrogen receptors in immune cells, this could stimulate the production of immunoglobulins (Kanda and Tamaki, 1999), increase the survival of B cell mediators (Cyster and Goodnow, 1995; Dinesh et al., 2010), or increase the number of regulator T cells (Arruvito et al., 2007).

CONCLUSION AND FUTURE DIRECTIONS

This study demonstrates that subacute exposure to DiNP increases proliferation, mucin production, and expression of Paneth cells in the colon. Furthermore, DiNP exposure significantly increases circulating estradiol levels compared with control. Future studies should focus on the role of goblet and Paneth cells in DiNP exposure models as such studies may provide information on host-mediated immune responses. Future studies should also examine the gut microbiome to see whether microbes contribute to the DiNP-induced effects on the colon.

SUPPLEMENTARY MATERIAL

Supplementary data are available at Toxicological Sciences online.

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, funding, and/or publication of this article.

Supplementary Material

kfab105_Supplementary_Data
Click here for additional data file. (15.2KB, docx)

ACKNOWLEDGMENTS

We sincerely thank Dr. Daryl D. Meling and Dr. Catheryne (Katie) Chiang for their assistance with the oral dosing studies. Lastly, we would like to acknowledge the Core Facilities at the Carl R. Woese Institute for Genomic Biology for equipment usage.

Funding

This work was supported by the National Institute of Health (NIH T32 ES007326 and R01 ES02866), the Division of Nutritional Sciences (Vision 20/20) at the University of Illinois at Urbana-Champaign, Environmental Toxicology Scholar Award, and the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign.

AUTHOR CONTRIBUTIONS

J.A.F. supervised the experimental work, edited the manuscript, and acquired funding for this study. K.C. designed and performed the experimental work and wrote the manuscript. S.T.B. and J.C. helped complete experimental work, conduct data analysis, and provided insights on the discussion section. R.A.N. provided critical insights into the study, helped with study design, and edited the manuscript.

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