Chronic arsenic exposure affects stromal cells and signaling in the small intestine in a sex-specific manner

Abstract

Arsenic is a toxicant that is ingested through drinking water and food, exposing nearly 140 million people to levels above the 10 ppb guideline concentration. Studies have shown that arsenic affects intestinal stem cells (ISCs), but the mechanisms by which arsenic alters the formation of adult cells in the small intestine are not well understood. Signals derived from intestinal stromal cells initiate and maintain differentiation. The goal of this study is to evaluate arsenic’s effect on intestinal stromal cells, including Pdgfrα^Lo trophocytes, located proximal to the ISCs, and Pdgfrα^Hi telocytes, located proximal to the transit-amplifying region and up the villi. Adult Sox9^{tm2Crm−EGFP} mice were exposed to 0, 33, and 100 ppb sodium arsenite in their drinking water for 13 weeks, and sections of duodenum were examined. Flow cytometry indicated that arsenic exposure dose-responsively reduced Sox9+ epithelial cells and trended toward increased Pdgfrα+ cells. The trophocyte marker, CD81, was reduced by 10-fold and 9.0-fold in the 100 ppb exposure group in male and female mice, respectively. Additionally, a significant 2.2- to 3.1-fold increase in Pdgfrα^Lo expression was found in male mice in trophocytes and Igfbp5+ cells. Pdgfrα^Hi protein expression, a telocyte marker, was more prevalent along the villus/crypt structure in females, whereas Gli1 expression (telocytes) was reduced in male mice exposed to arsenic. Principle coordinate analysis confirmed the sex-dependent response to arsenic exposure, with an increase in trophocyte and decrease in telocyte marker expression observed in male mice. These results imply that arsenic alters intestinal mesenchymal cells in a sex-dependent manner.

Arsenic is a prevalent toxicant found to affect between 94 and 220 million people in 50 countries who are exposed to levels above the acceptable guideline concentration of 10 μg/l (or 10 ppb) (Podgorski and Berg, 2020). The primary route of arsenic exposure is through the consumption of contaminated drinking water from areas in which arsenic is highly bioavailable, such as parts of China, Bangladesh, and the western United States (Calatayud et al., 2015). In addition, crops grown in these regions, such as rice, can bioaccumulate arsenic resulting in another source of exposure. Chronic exposure to arsenic above the drinking water limit can result in cancer, skin lesions, an increased risk of cardiovascular disease and diabetes, and decreased cognitive development in young children (Raju, 2022; Zheng et al., 2021). Additionally, evidence suggests it can also act to impair the differentiation of muscle and neuronal progenitor cells as well as of stem cells (Cheikhi et al., 2020; Liu et al., 2012; McMichael et al., 2021). The small intestine relies on stem cells to maintain a steady supply of rapidly differentiating epithelial cells. Because it is also the primary route of absorption and has the tissue with the highest concentrations of arsenic following oral exposure (El-Masri et al., 2018), the small intestine presents itself as a potential target of arsenic’s toxicity.

The small intestine plays a vital role in absorbing nutrients. Every 5–7 days, the intestinal epithelium regenerates itself, with each of the different cell types (enterocytes, tuft, goblet, enteroendocrine, and Paneth cells) arising from intestinal stem cells (ISCs) (McCarthy et al., 2020; Shroyer et al., 2005; Stappenbeck, 2009). The general structure of the small intestine includes epithelial protrusions called villi and epithelial cell pockets called crypts, which contain the ISCs. Behind the single cell thick epithelial layer, a sublevel of several stromal cell types exists. Maintenance of intestinal regeneration and differentiation is dependent upon a balance between Wnt signal isoforms localized around the crypt preserving stemness and Bmp isoforms in the villus maintaining region of differentiation (McCarthy et al., 2020). Other key signaling factors include those in the Hedgehog, epithelial growth factor (EGF), notch, hippo, and map-kinase pathways (Farin et al., 2012; McCarthy et al., 2020). These signaling molecules typically are secreted from the underlying stromal cells, and thus it is the stromal cells that are responsible for maintaining the stem cell niche, regulating appropriate cell differentiation, and responding to damage (Greicius and Virshup, 2019; McCarthy et al., 2020; Owens and Simmons, 2013; Wu et al., 2021). Stromal cells can be divided into 2 main categories: Pdgfrα expressing and nonexpressing cells. Within the Pdgfrα expressing cell category, there are telocytes (Pdgfrα^Hi, CD201, Gli1), trophocytes (Pdgfrα^Lo, CD81+, Grem1+), Fgfr2+ fibroblasts (Pdgfrα^Lo, CD81−, Grem1−), and Igfbp5+ fibroblasts (Pdgfrα^Lo, CD81−, Grem1+). Telocytes express much higher levels of Pdgfrα (Pdgfrα^Hi) and are primarily Bmp producers with the ability to secrete noncanonical Wnt4 and 5a/b (Bahar Halpern et al., 2020). Trophocytes are Pdgfrα^Lo cells located just below the crypt and ISCs, and these secrete Gremlin1, Rspo, and canonical Wnt2b/9a (Farin et al., 2012; Flanagan et al., 2018; Hendel et al., 2022; Kaestner, 2019; Lee et al., 2020; McCarthy et al., 2020; Worthington et al., 2017). Igfbp5+ fibroblasts are thought to assist in ISC niche maintenance, whereas the role and function of Fgfr2+ fibroblasts are relatively unknown other than its link to suppression of trophocyte-induced crypt expansion (McCarthy et al., 2020).

Because the main route of arsenic exposure is ingestion via drinking water and food, the small intestine is likely a target for arsenic. There have been some studies that suggest arsenic impairs the differentiation of small intestine epithelial cells. A study observing the effects of As(III) on the small and large intestine found increased reactive oxygen species and lipid peroxidation alongside increased pro-inflammatory cytokines (Domene et al., 2023). Arsenic affects the gut microbial alpha diversity or species richness; however, the populations did not vary in beta diversity, which measures the differences between exposure groups (Domene et al., 2023; Li et al., 2021). Further, research has shown that arsenic directly impacts gut permeability and tight junctions, as ZO-1 and claudin levels are reduced by cytokine release (IL-8 and TNF-α) (Chiocchetti et al., 2018; Zhong et al., 2021). In a 5-week exposure study, mice exposed to 100 ppb sodium arsenite in their drinking water had reduced Lgr5 expression, a marker of ISCs, along with decreases in markers of secretory cells such as Defa1, Tff3, and Atoh1 (Jatko et al., 2021). Additionally, arsenic decreased the width of the lamina propria, reduced Pdgfrα expression by 4.2-fold, and reduced Grem1, Gli1, and Cd81 gene expression (Kellett et al., 2022). Linear regression models from this study suggested that a 5-week arsenic exposure affected stromal cells of the small intestine (Kellett et al., 2022).

Overall, the purpose of this study is to examine whether arsenic exposure alters the numbers, localization, and/or function of stromal cells within the small intestine. Mice were exposed to 33 and 100 ppb arsenic for 13 weeks via drinking water, and changes in intestinal stromal and epithelial cells were examined using flow cytometry, immunohistochemistry, and qPCR. Sex-specific responses to the arsenic exposures were observed, with males exposed to arsenic having increased expression of trophocyte markers, whereas females exposed to arsenic had increased expression of telocyte markers.

Materials and methods

 

 

Mouse exposure to arsenic

A total of 36 Sox9^tm2Crm-EGFP mice (strain B6:129S4-Sox9<tm1.1Tlu>/J; Jackson Laboratories; Bar Harbor, Maine; 18 males and 18 females) were divided into 3 groups at 8 weeks of age and exposed to 0, 33, and 100 ppb arsenic (as sodium arsenite, Fisher Scientific; Hampton, New Hampshire) in their drinking water for 13 weeks (n = 6 mice per sex). These transgenic Sox9-EGFP mice have been used to assess different intestinal cell populations based upon fluorescent expression (Van Landeghem et al., 2012). Mice are known to have greater resistance to arsenic toxicity than humans due to differences in the AS3MT gene, which is responsible for methylation of arsenic metabolites into monomethyl- and dimethyl-arsenic acid (Stýblo et al., 2019, 2021; Vahter and Concha, 2001). However, there are no differences in As3mt gene expression between sexes in mice. The equivalents of 33 and 100 ppb in a human exposure would be approximately 130 and 400 ppb, based on liver concentrations measured at 25 ppb inorganic arsenic (iAs) exposure (Douillet et al., 2023).

The drinking water was changed 3 times per week, and mice were weighed weekly. Drinking water and rodent chow (AIN-93G, Envigo; Indianapolis, Indiana) were analyzed for arsenic levels, as well as other trace elements, by ICP (Thermo Fisher iCAP 7200 ICP-OES Analyzer) using Clemson’s Agricultural Service Center. There were no detectable levels of arsenic in the tap water, nor were there any changes in arsenic concentrations in the water spiked with arsenic after 72 h. Thirty minutes prior to euthanasia, mice were gavaged with 50 mg/kg FD-4 (fluorescein isothiocyanate dextran-4, Sigma-Aldrich; St Louis, Missouri) dissolved in approximately 100 µl sterile water to assess intestinal barrier integrity. Mice were euthanized, and blood collected for serum preparation. A 10-cm section of small intestine adjacent to the stomach (duodenum) was collected and flushed with cold PBS before being subdivided into small (approximately 1 cm) pieces. The duodenum was selected due to being the first section of the small intestine capable of absorbing phosphate, which shares chemical characteristics with sodium arsenite (Knodle et al., 2012; Sabbagh et al., 2011). Two pieces were fixed in 4% formalin for use in IHC, 2 pieces were placed into Tri-Reagent (Sigma-Aldrich; St Louis, Missouri) for qPCR analysis, and 5–7 pieces were put into Dispase (Roche; Basel, Switzerland) and collagenase (Sigma-Aldrich; St Louis, Missouri) for use in flow cytometry.

Flow cytometry

To analyze specific cell populations, flow cytometry was conducted. The pieces of small intestine in Dispase/collagenase solution were incubated at 37°C for 5–10 min to obtain single cell suspensions before being diluted and resuspended in FACS buffer (1× PBS, 1% BSA, 0.1% sodium azide; 1 × 10⁶ cells/ml). The first sort separated epithelial cells from stromal cells using an E-cadherin primary antibody (rabbit mAb, no.3195, Cell Signaling Technologies; Danvers, Massachusetts, 1:200) and a secondary Alexa Fluor 568 nm antibody (Invitrogen, Waltham, Massachusetts, 1:200), each of which were incubated for 30 min at 4°C. Cells were washed 3 times in FACS buffer and preserved in 4% paraformaldehyde. The expression of Sox9-EGFP in specific small intestinal epithelial cells from the transgenic mice was assessed at 488 nm. The negative control was nontagged small intestine single cell suspension to define Sox-9-positive/negative cells. Sox9 subregions were defined based on the Sox9-positive control into Sox9-Hi (>10³ RFU), Sox9-Lo (500–1000 RFU), and Sub-Lo (158–1000 RFU) populations, which are indicative of enteroendocrine cells, ISC’s and transit amplifying cells, respectively (Gracz et al., 2010; Van Landeghem et al., 2012).

The unstained nonepithelial cells underwent a second sort into Pdgfrα stromal cells and CD45 immune cells, using antibodies to CD45 tagged to Alexa Fluor 561 (no.505-0451-82, ThermoFisher; Waltham, Massachusetts 1:200) and Pdgfrα (no. sc-21789, Santa Cruz Biotechnology; Dallas, Texas, 1:200) and a secondary Alexa Fluor 488 nm antibody (Invitrogen, 1:200) for 30 min. Cell populations were analyzed by defining gates based on negative controls in FloJo to determine the total number of positive cells for each marker tested and the percentage of the total cell population.

Quantitative PCR

RNA was extracted from intestinal samples, and concentration and purity determined using a NanoDrop spectrophotometer (ThermoFisher). RNA (2 µg) was reverse transcribed into cDNA using MMLV reverse transcriptase (Promega; Madison, Wisconsin). Primers were validated using PCR, and then purified using MiniElute PCR purification kit (Qiagen; Germantown, Maryland). The purified PCR product of each gene was used to create a standard curve. Gene expression was analyzed on a Bio-Rad iQ5 Thermocycler (Hercules; California) using RT² SYBR Green (Applied Biosystems; Foster City, California) with specific gene targeting primers (Supplementary Table 1). A standard curve of purified PCR product at concentrations from 10⁻³ through 10⁻⁷ng cDNA were run in triplicate determine run efficiency. Data were normalized with housekeeping genes Gapdh and β2-microglobulin using the Pfaffl method (Pfaffl, 2001).

Paraffin embedding and Masson’s trichrome stain

Intestinal samples were fixed in 4% formalin, dehydrated, and embedded in paraffin. Blocks were sectioned using a microtome (8–10 μm), which were placed onto positively charged slides. Masson’s trichrome staining was conducted according to manufacturer’s instructions (Polysciences Inc., Warrington, Pennsylvania). Images were captured using a Leica DMi1 (Leica; Wetzlar, Germany) with a 10× objective (Leica; Wetzlar, Germany). Collagen width was assessed by taking 3 width measurements below the crypt of the blue stained collagen (n = 4–6 mice per group).

Immunohistochemistry

Paraffin-embedded intestinal samples were sectioned (10 µm) with a microtome, placed on slides, and rehydrated through a graded ethanol solution. Antigen retrieval was performed using citric acid buffer, pH 6 or Tris-EDTA buffer, pH 9. Samples were blocked in 5% BSA/PBST. Before IHC was performed on all samples, the appropriate antigen retrieval solution and antibody concentrations were determined (Supplementary Table 2). Primary antibody was incubated overnight at 4°C, washed 3 times with 5% BSA in PBST, incubated with the secondary antibody for 1 h at room temperature, and washed with 5% BSA in PBST. The secondary antibodies used are Alexa Fluor 488, Alexa Fluor 555, 568, or 594, and Alexa Fluor 647 (Invitrogen; 1:500–1:1000). TOPRO-3 (Invitrogen; 1:1000) was used in some procedures to counterstain the nuclei. Sections were imaged on a Leica DM2500 Confocal microscope (Leica Microsystems, Buffalo Grove, Illinois) with 488, 532, and 635 nm lasers. Samples were imaged using either a 20× objective (NA = 0.75) or 63× objective (NA = 1.4) with a zoom of 1.0. Additionally, parameters defined on the confocal remained consistent throughout the imaging process for the corresponding slide set (n = 18) with 3 images taken of each slide at the desired magnification, either 20× for observing of the villus or 63× for focused observation on crypt markers (Supplementary Table 2).

Statistical analysis

Mice were weighed weekly and the change in weight over time was determined by subtracting the week 0 weight. Data were averaged by sex and exposure group (n = 6). Collagen width was measured on intestinal sections stained with Masson trichrome and averaged per sex and exposure group (n = 4–6). Data were analyzed in GraphPad using ANOVA with Tukey’s. The flow cytometry data were grouped by exposure (0, 33, and 100 ppb) then concatenated into a single data file by exposure group. Gates were defined based on positive controls for each marker (Sox9, Pdgfrα, and Cd45) at 60 RFUs. To determine percentage of cell count for Pdgfrα and Cd45 as the second sort, the percentage of Pdgfrα and Cd45 positive cells was multiplied by first sort percentage of Sox9⁻/E-cad⁻ cells. The cell count percentages were averaged per exposure group and sex. For analysis of qPCR data, the geometric mean of Gapdh and β2-microglobulin were used as housekeepers, and the Pfaffl method was used to quantify gene expression changes (n = 4–6 mice per sex per exposure group). For analysis of IHC, FIJI was used to assess changes in cell count and fluorescent intensity of 4K resolution images (n = 4–6 mice per sex; 3 images per mouse). To assess the number of neutrophils and macrophages, Ly-6C/G and F4/80 expression regions that had cell-like structure (Supplementary Figure 2) were counted within the villus and cryptal regions of the lamina propria (n = 2–6) then standardized by the total number of villi. Further, the integrated density (area selected times mean gray value) was determined by measuring pericrypt and crypt regions for Pdgfrα^Lo and Grem1, while measuring dimensions of expression and expression intensity for Pdgfrα^Hi and α-SMA. Additionally, cell signaling gradients along the villus were assessed by measuring the length of expression standardized to the villus length of IHC images with 3 villi per sample (n = 5 mice per sex) for α-SMA and Pdgfrα. For male, female, and combined datasets, the mean and standard deviations were calculated. Statistical differences were assessed by parametric ANOVA, followed by Tukey’s and Dunnett’s on Graphpad Prism 9 (San Diego, California). Principle coordinate analysis (PCA) for each individual mouse was conducted with the stromal related data in GraphPad Prism 9.

Results

A total of 36 Sox9^tm2Crm-EGFP mice (18 male and 18 female) were exposed to 0, 33, and 100 ppb in their drinking water for 13 weeks. No morbidity or mortality was seen throughout the course of the experiment. Male and female weight gain recorded over 13 weeks found that, on average, male mice gained 14 g, whereas female mice gained 6 g. However, there were no differences in weight gain between exposure groups (Table 1).

Table 1.

Weight gain was not different between the exposure groups after 13 weeks of arsenic exposure

	Weight gain (g)
	0 ppb As	33 ppb As	100 ppb As
Males	12.2 ± 2.9	15.8 ± 3.2	12.3 ± 1.9
Females	5.8 ± 1.5	6.8 ± 2.0	5.6 ± 1.9

n = 6 mice per exposure group.

 

Exposure to arsenic increased neutrophil cell count of male mice

To assess the localized immune response within the small intestine, macrophage and neutrophil cell counts were assessed using IHC with Ly-6C/G (neutrophils) and F4/80 (macrophage) antibodies. Although neutrophil cell count was significantly increased in male mice at 100 ppb from an average of 1 neutrophil per villus to 4 neutrophils per villus (Figure 1B); however, the total number of macrophages did not significantly increase in males (Figure 1A). Additionally, a nonsignificant dose-response increase was observed in females for both macrophages and neutrophils (Figs. 1A and 1B).

Figure 1.

Arsenic increased neutrophil cell counts in male mice at 100 ppb. Macrophage (A) and neutrophil (B) cell counts were determined following immunohistochemistry with Lys6 and F4-80 antibodies. Statistical differences were determined by ANOVA followed by Tukey’s (*p < .05; n = 2–6).

Arsenic exposure decreased collagen width in the small intestine of male mice

Because previous studies had shown a reduction in collagen levels in male mice exposed for 5 weeks to 100 ppb arsenic (Kellett et al., 2022), we also quantified collagen levels in the lamina propia of each mouse following Masson’s trichrome stain (Figs. 2A and 2C). Although no obvious morphological differences were seen between exposure groups, a dose-responsive reduction in lamina propia collagen width was observed in male mice, which was statistically significant in 100 ppb exposure group (Figure 2B). In females, changes in collagen width were not observed (Figure 2D). One of the main cell types that secretes collagen in the small intestine are myofibroblasts, which can be assessed using IHC to examine the expression of α-smooth muscle actin (α-SMA) (Mifflin et al., 2011). In the area below the crypts, α-SMA width did not change in either males or females (Supplementary Figure 4). In the villus, the length of α-SMA expression was normalized for the total length of the villus (Figure 3A). No significant differences were found in males or females alone, nor when combining the 2 sexes together (Figure 3B). Next, the intensity of α-SMA expression in the villi was analyzed. In male mice, no changes in α-SMA intensity were found (Figs. 3C and 3D). In female mice, α-SMA intensity in the villus has a significant dose-responsive reduction (p = .02; Figs. 3E and 3F).

Figure 2.

Arsenic reduced collagen width in the lamina propia in male mice. Masson trichrome stain was used on sections of small intestine and imaged on DMi1 microscope using Flexacam C1 (A, B: blue = collagen; purple = cytoplasm; black = nuclei). Collagen width was then analyzed using ImageJ with 3 measurements per mouse (yellow bracket) from crypt to smooth muscle layer (C: n = 4–6). Scale bar = 75 μm. Collagen width was averaged per exposure group and statistical differences determined by ANOVA followed by Tukey’s (*p < .05; n = 6 mice per exposure group).

Figure 3.

Arsenic does not alter α-SMA protein expression in the villus. Immunohistochemistry was performed using antibodies to α-SMA (red) and EGFP (green). TOPRO-3 (yellow) was used to counterstain nuclei. Images were captured with the Leica LAS X package, and Image J used to measure villus length (blue arrow) and α-SMA length (yellow arrow) (A). Then, the percentage of α-SMA length along each villus was determined (B). To assess α-SMA (red) intensity in the villus, α-SMA-only 3D images for males (C) and females (E) were analyzed in ImageJ and quantified as an integrated intensity. Data are presented as the average and standard deviation (D and F). Scale bar = 100 μm. Statistical differences were determined using ANOVA followed by Tukey’s and Dunnett’s (*p < .05; n = 4–6 replicates per sex per exposure group).

Arsenic exposure alters epithelial cell populations as evidenced by flow cytometry

Following euthanasia, the small intestine was removed and sectioned to use for flow cytometry, qPCR, and histochemistry. First, flow cytometry was used to assess specific epithelial and stromal cell numbers. Sox9 is expressed in transit amplifying cells, ISCs, and enteroendocrine cells, so its expression was first assessed. Exposure to 33 and 100 ppb arsenic caused a distinct reduction in the percentage of cells that were Sox9+ (Figure 4A and Supplementary Figure 1). The percentage of Sox9+ cells decreased from 50.5% in male control mice to 39.5% in male mice exposed to 100 ppb arsenic, whereas percentages went from 63% in female control mice to 44% in female mice exposed to 100 ppb arsenic (p = .06; Supplementary Table 3). An overall decrease in Sox9+ cells indicates that arsenic may be impairing differentiation, so the Sox9+ cells were divided into 3 distinct subgroups according to their relative Sox9 fluorescence levels: transit amplifying cells (Sox9^Sublo), ISCs (Sox9^Lo), and enteroendocrine cells (Sox9^Hi) (Figure 4B) (Gracz et al., 2010, 2012). Although the transit amplifying region of the intestine, marked by Sox9^Sublo, appeared to decrease due to arsenic exposure, this was not statistically significant (Figure 4B and Supplementary Table 3). Additionally, there were no changes in the percentage of ISCs, marked by Sox9^Lo, nor in enteroendocrine cells, marked by Sox9^Hi (Figure 4B). Cells that were Sox9⁻ underwent a second sort to assess the numbers of stromal cells (Pdgfrα+) and immune cells (CD45+). The results revealed no changes in the percentage of either Pdgfrα+ cells (Figure 4C) or CD45+ cells (Figure 4D and Supplementary Table 3).

Figure 4.

Flow cytometry indicates that arsenic exposure altered cell populations. In the first sort, Sox9 and E-cadherin expression were used to determine the number of epithelial cells expressing Sox9 (marker for transit amplifying, intestinal stem, and enteroendocrine cells). The data are shown as number of Sox9⁺/E-cadherin⁺ cells out of 20 000 total cells counted per mouse (A: n = 10–12 per exposure group). To determine Sox9 subpopulations, 3 regions were defined and gated in FloJo software: Sox9^Sublo (transit amplifying cells), Sox9^Lo (intestinal stem cells), and Sox9^Hi (enteroendocrine cells). Data shown in the average cell counts for each exposure group (n = 10–12 mice) (B). The nonepithelial cell population was further sorted to determine stromal cell counts using antibodies to Pdgfrα (C) and immune cell counts using antibodies to CD45 (D). These counts were then converted to a percentage by dividing the Pdgfrα (+) and CD45 (+) cell count by the total cell count of 10 000 (C and D). Statistical differences were determined by ANOVA followed by Tukey’s (*p < .05; n = 10–12 mice per exposure group).

Arsenic exposure altered trophocyte and telocyte mRNA markers in a sex-dependent manner

Because a 13-week arsenic exposure appeared to reduce the number of Sox9+ cells while concomitantly increasing the number of Pdgfα+ stromal cells, markers of specific stromal cells were next examined. The small intestine has several major stromal cell types, including trophocytes (marked by Pdgfrα, Cd81, and Grem1), and telocytes (marked by Pdgfrα, CD201, and Gli1). qPCR analysis of Pdgfrα gene expression, as a marker for both telocytes and trophocytes, was not changed in either males or females (Figs. 5A and 5F). CD201, a telocyte marker, increased in a dose-dependent manner in males (Figure 5B) and females (Figure 5G), although these changes were not statistically significant. Interestingly, the telocyte marker Gli1 was decreased in males by 2.0-fold and 1.7-fold in the 33 and 100 ppb exposure groups (Figure 5C), whereas in females, no changes in expression were seen (Figure 5H). The trophocyte marker CD81 was significantly decreased in male mice, being reduced by 1.4-fold in 33 ppb exposure group and 10-fold in the 100 ppb exposure group (Figure 5D), whereas in female mice, a 9.0-fold decrease was observed (Figure 5I). In contrast, Grem1 expression remained unchanged in both male (Figure 5E) and female mice (Figure 5J).

Figure 5.

Arsenic exposure impairs trophocyte markers in male and female mice, and telocyte markers in male mice. Relative transcript expression levels of stromal cell markers were assessed by qPCR. Transcripts examined included Pdgfrα (A and F), the telocyte markers CD201 (B and G), and Gli1 (C and H), and the trophocyte markers CD81 (D and I) and Grem1 (E, J). Fold change in male and female mice exposed to 33 ppb (orange) and 100 ppb (red) arsenic was determined using the Pfaffl method and normalized to geometric mean of Gapdh and β2-microglobulin. Male samples are indicated with a triangle (▲) and female samples are indicated with a circle (●). Statistical differences were determined using ANOVA followed by Tukey’s and Dunnett’s (*p < .05; n = 4–6).

Sex-dependent differences in Pdgfrα protein expression due to arsenic exposure

Pdgfrα is expressed in several cell types. In the pericryptal region, trophocytes typically express Pdgfrα at lower intensities (Pdgfrα^Lo), whereas in the villus, telocytes express it highly (Pdgfrα^Hi) (Koliaraki and Kollias, 2016). Additionally, Igfbp5+ fibroblasts in the crypt itself also express Pdgfrα^Lo (McCarthy et al., 2020; Pærregaard et al., 2023). Thus, Pdgfrα protein expression was assessed using immunohistochemistry to focus on these 3 distinct cell types. Pdgfrα^Lo expression within the crypt was significantly increased in males by 2.7-fold and 3.1-fold at 33 and 100 ppb, respectively (Figs. 6A and 6C). In addition, Pdgfrα^Lo expression within the pericryptal region was altered at 100 ppb significantly by 3.0-fold (Figs. 6A and 6C). Although in females, the opposite trend was found with a decrease in Pdgfrα^Lo expression within the crypt region by approximately 10-fold from corresponding female controls, whereas this was not significant (Figs. 6D and 6F). There were no sex differences in Pdgfrα^Lo crypt or pericrypt intensity between control males and females (p = .09 and .22, respectively). To assess Pdgfrα^Hi expression, both intensity of expression and then length of that expression were measured. In both males and females, overall Pdgfrα^Hi intensity was not significantly altered (Supplementary Figure 4). Further, the length of Pdgfrα^Hi expression along the villus was not altered in males (Figs. 6B and 6C). However, the length of Pdgfrα^Hi expression in female mice was increased in a dose-responsive manner. In the controls, Pdgfrα^Hi expression extended to 40% of the villus, whereas in the arsenic-exposed mice, expression extended to 47% and 53% of the villus in the 33 and 100 ppb exposure groups, respectively (Figs. 6E and 6F). These effects suggest that arsenic exposure alters male trophocytes and female telocytes in a sex-dependent manner.

Figure 6.

Arsenic exposure increased Pdgfra^Lo expression in males and increased Pdgfra^Hi expression along the villus in females. Immunohistochemistry was used to analyze Pdgfrα^Lo expression within the crypt region (light orange) and pericryptal region (red dashed area) (A and D). Additionally, Pdgfra^Hi expression was assessed within the villus region by measuring length of expression (pink arrow) to villus length (blue arrow) (B and E). Markers present in Pdgfrα^Lo expression analysis are Sox9-EGFP (green), Pdgfrα (red), and Olfm4 (yellow), for which analysis of integrated intensity was performed in ImageJ (A and D). Pdgfra^Hi expression was analyzed by measuring its length of expression (pink arrow) and comparing this measurement to total villus length (blue arrow) (B and E). Scale bar = 40 μm (A and D); scale bar = 100 μm (B and E). Statistical differences were determined using ANOVA followed by Tukey’s and Dunnett’s (C and F: *p < .05; **p < .01; ***p < .001; n = 4–6).

Grem1 expression was altered in a sex-dependent manner

Grem1 is a key cell signaling protein within the ISC niche that is secreted by trophocytes to prevent premature differentiation of stem cells (McCarthy et al., 2020). Because sex-dependent changes in the protein expression of the trophocyte and Igfbp5+ marker Pdgfrα^Lo were found in the pericryptal and cryptal region respectively, Grem1 expression was also assessed to determine if these changes persisted in the signaling protein produced by these cell types (McCarthy et al., 2020; Pærregaard et al., 2023). Within the cryptal region, male mice exposed to 33 or 100 ppb arsenic had a 2.4- and 2.0-fold increase in Grem1 protein expression within the pericryptal region, respectively, which was significant at 100 ppb (p = .04) (Figs. 7A and 7B). Grem1 expression within the pericryptal region was not altered significantly. In females, there were no differences in Grem1 expression (Figs. 7D and 7E). Because both trophocytes and Igfbp5+ fibroblasts secrete Grem1, linear regression between Grem1 and Pdgfrα^Lo protein expression was conducted. In males, Grem1 and Pdgfrα^Lo levels within the crypt were significantly correlated, with control mice having relatively low expression of both proteins (green triangles), whereas arsenic-exposed mice (orange and red triangles) had high expression of both proteins (p = .05) (Figure 7C).

Figure 7.

Grem1 protein expression in the crypt. Immunohistochemistry was used to analyze Grem1 expression (red) within the crypt (pink circle) and around its perimeter (red dashed area) of the small intestine in both male (A) and female (D) Sox9^EGFP mice (green) exposed to 0, 33, or 100 ppb arsenic. ImageJ was used to determine the expression of Grem1 by integrated density (B, D, and E). Linear regression of Grem1 expression to Pdgfrα^Lo expression (C). Scale bar = 40 µm. ANOVA followed by Tukey’s and Dunnett’s was used to determine statistical significance between exposure groups (*p < .05; n = 4–6).

Population shift in arsenic-exposed males toward trophocyte markers

Because sex-dependent changes were seen in several stromal markers due to arsenic exposure, PCA was used to examine these relationships. The model contained data for trophocytes (Pdgfrα^Lo protein expression, Pdgfrα, Grem1, and CD81 mRNA expression) and telocytes (Pdgfrα^Hi protein expression, Pdgfrα length, Pdgfrα, Gli1, and CD201 mRNA expression), α-SMA width and length, and Cd45 and Pdgfra flow cytometry population percentages. The resultant biplot shows that both male and female control mice are grouped together in the upper right quadrant (green circle; Figure 8). The arsenic exposure groups shift away from the controls in a sex-dependent manner. The male 33 ppb exposure group (dark orange circle; Figure 8) and male 100 ppb exposure group (dark red circle; Figure 8) shifted to the left 2 quadrants, which is indicative of increasing levels of the trophocyte marker Pdgfrα^Lo and decreasing levels of the telocyte marker Gli1. In contrast, the female exposure groups are located in the right 2 quadrants (Figure 8). In particular, the 33 ppb females shifted slightly to the lower right corner, whereas telocyte markers (Gli1, CD201, and Pdgfrα^Hi protein expression and length) are located (light orange circle; Figure 8). A shared commonality between the sexes was CD81 expression, which was decreased at 100 ppb in males and females. Overall, the variance seems to be driven by 100 ppb exposure along the PC1 axis, with sex differences observed along the PC2 axis. With PC1 and PC2, the cumulative proportion of variance is 73% (Figure 8).

Figure 8.

Principle coordinate analysis (PCA) shows differences between arsenic exposure groups and genders. A biplot was constructed from PCA using all the stromal measurements (transcripts, protein) for each individual mouse (n = 2–5 per exposure group per sex). The green circles surround the region in which male and female controls were grouped as 2 distinct populations. Light orange surrounds the female 33 ppb exposure and dark orange the 33 ppb males. Light red surrounds the location of the 100 ppb females and dark red the 100 ppb males.

Discussion

These results suggest that a chronic 13-week exposure to 100 ppb arsenic in drinking water results in sex-specific changes in the stromal cells of the small intestine. In males, markers indicative of trophocytes, such as Pdgfrα^Lo, were increased. In contrast, females exposed to arsenic has increased markers of telocytes, such as the increased expression of Pdgfrα^Hi along the villus.

 

Arsenic altered trophocyte markers in males

In the male mice exposed to arsenic, IHC revealed a significant increase in Pdgfrα^Lo cells in the small intestine. These specific stromal cells can be further divided into 3 cell types. Trophocytes (Pdgfrα^Lo/CD81/Grem1) are located proximally to the crypt (Hong et al., 2020; McCarthy et al., 2020) and help maintain the ISCs niche by producing Grem1 and canonical Wnt2b/9a (Farin et al., 2012). IHC analysis demonstrates that a chronic 13-week 100 ppb arsenic exposure increases the number of Pdgfrα^Lo cells. Similarly, Grem1 mRNA levels also appear to be increased in the male mice. Interestingly, these changes in Grem1 were not observed in a previous study when male mice were exposed for only 5 weeks to 100 ppb arsenic (Kellett et al., 2022). But, given the differences in exposure times (5 weeks vs 13 weeks), these differences might not be surprising. Indeed, Paneth cells in the small intestine can live for up to 8 weeks (Ireland et al., 2005), and it typically takes 2–3 months for intestinal crypts to become monoclonal (Snippert et al., 2010).

In contrast, CD81 gene expression was significantly decreased in both male and female mice at 100 ppb in the current 13-week study, as well as in a previous male-only 5-week 100 ppb arsenic exposure study (Kellett et al., 2022). CD81 is part of the family of transmembrane proteins known as tetraspanins (Lang and Hochheimer, 2020). Why its expression is declining in the arsenic-exposed mice in unknown. However, in addition to being a marker of trophocytes, CD81 regulates cell migration through its interaction with α3β1 integrin (Yáñez-Mó et al., 2021; Bailly and Thuru, 2023). Several studies have found that arsenite exposure increases the expression of multiple integrin subunits, including β1 integrin (Ji et al., 2019; Zhang et al., 2023), but these studies examined changes in arsenic-induced cell migration in cancer cells.

There are 2 other subtypes of Pdgfrα^Lo cells (also known as Lo-2) that have recently been identified. These subpopulations are described as being either Igfbp5+ or Fgfr2+ cells (Brügger and Basler, 2023; Chalkidi et al., 2022). The Pdgfrα^Lo Fgfra2+ cell population, which is located in the villus interstitium and highly expresses genes linked to epithelial support and differentiation, along with immune response (McCarthy et al., 2020; Pærregaard et al., 2023). They also appear to secrete a variety of Bmp isoforms (Brügger and Basler, 2023).

The second subtype are the Igfbp5+ Pdgfrα^Lo CD81- Grem1+ fibroblasts, which are located in the crypt (Brügger and Basler, 2023). These cells secrete proteins that maintain the intestinal cell niche, such as those in the Wnt pathway, and proteins that protect against premature differentiation, such as the Bmp antagonist Grem1. These cells also secrete basement membrane proteins and construct the extracellular matrix (Pærregaard et al., 2023). In the crypt, Grem1 protein expression is significantly increased in the 100-ppb male exposure group. Additionally, a significant correlation exists between Pdgfrα^Lo and Grem1 expression in the crypt. Taken together, our data indicate that chronic arsenic exposure increases both trophocyte and Pdgfrα^Lo Igfbp5+ cells in the small intestine of the males, thereby increasing the expression of the Bmp antagonist Grem1.

Arsenic affects telocytes in female mice

Unlike the males, arsenic exposure to female mice had little effect on the small intestinal stromal cells, except for an increased presence of telocytes along the villus as indicated by Pdgfrα^Hi. Telocytes are responsible for promoting the continued differentiation and migration of epithelial cells from the transit amplifying region to the villus by expressing signaling molecules such as noncanonical Wnts, BMP, and Lgr5+ (Bahar Halpern et al., 2020; McCarthy et al., 2020). Another marker of telocytes is CD201. Although CD201 mRNA expression in the females appears to be increased by approximately 3-fold in the arsenic-exposed mice, this was not statistically significant. Additionally, linear regression between CD201 mRNA and Pdgfra^Hi protein expression also revealed a correlation trend, one that would indicate a higher level of expression in the female arsenic-exposed mice, but this trend was also not statistically significant (p = .1; Supplementary Figure 5). Interestingly, the trophocyte marker CD81 was significantly reduced, similar to what was seen in the males.

A previous study demonstrated that a 5-week exposure to 100 ppb arsenic altered Defa1, Tff3, and Atoh1 mRNA expression, all markers of secretory epithelial cells in the small intestine (Jatko et al., 2021). Based upon concurrent changes of Bmp4 and Grem1 mRNA, it was hypothesized that arsenic exposure might affect stromal cell signaling (Kellett et al., 2022). The current study investigated this hypothesis using a longer exposure period (13 weeks), 2 different arsenic concentrations (33 and 100 ppb), and examined stromal cell changes in the small intestine of both males and females. The results confirm stromal cell changes, demonstrating that male mice exposed to arsenic have increased expression of Pdgfrα^Lo CD81+ trophocytes, and Pdgfrα^Lo CD81− Igfbp5+ fibroblasts in the small intestine (Figure 9). In contrast, female mice exposed to arsenic have increased expression of the telocyte markers Pdgfrα^Hi CD201+ telocytes (Figure 9). Similarly, collagen levels in the lamina propia were reduced in male mice in both the previous 5-week study (Kellett et al., 2022) and the current 13-week study. In female mice, collagen expression was not reduced. There are limitations to colorimetric staining which may have precluded our ability to visualize more subtle changes in the females. However, α-SMA protein expression, a marker for myofibroblast stromal cells, was examined by IHC in which arsenic-exposed male and female mice show a trend toward reduced α-SMA levels. Further, there are other cells within the lamina propia that can secrete collagen in addition to myofibroblasts, such as Igfbp5+ fibroblasts and Fgfr2+ fibroblasts (McCarthy et al., 2020). One possibility for the sex differences may be due to changes in numbers and/or locations of these other cell types. However, we have been unsuccessful with IHC attempts to directly label these other fibroblast subtypes.

Figure 9.

Chronic arsenic alters stromal cells in the small intestine in a sex-dependent manner. Blue arrows represent significant changes observed in males, whereas red arrows represent significant changes observed in females.

The mechanisms driving sex differences in stromal cells are not entirely clear. A recent study demonstrated that iAs tissue concentrations do not vary between male and female mice, nor are there different proportions of methylated arsenical metabolites (Douillet et al, 2023). The PCA revealed a slight difference in grouping between control males and control females, which appears to be driven by changes in Pdgfra^Lo expression in the crypt and pericryptal area. However, taken alone, sex-dependent Pdgfra^Lo expression levels were not statistically significant. Considering that 33 and 100 ppb arsenic are a relatively low exposure concentration in mice, chronic arsenic exposure appears to be amplifying small changes in intestinal stromal cells between the sexes, resulting in sex-specific responses. Females exposed to arsenic had increased expression of telocyte markers, whereas arsenic-exposed males have increased expression of trophocyte markers.

Supplementary data

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, and/or publication of this article.

Funding

National Institute of Environmental Health Sciences (ES031766); a 2022 Biological Sciences Graduate Professional Development Research Grant.

Author contributions

Scott W. Ventrello (Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft, Writing—review & editing, Visualization), Nicholas R. McMurray (Validation, Investigation, Data curation), Nicholas M. Edwards (Validation, Investigation), and Lisa J. Bain (Conceptualization, Supervision, Validation, Formal analysis, Writing—review & editing, Visualization, Funding acquisition).