Detergent-containing toothpaste decreases esophageal mucosal impedance and alters salivary properties in humans

Abstract

Background:

Eosinophilic esophagitis is associated with epithelial barrier dysfunction. Epidemiologic studies suggest environmental factors promote disease pathogenesis. The common household detergent sodium dodecyl sulfate (SDS) induces epithelial barrier dysfunction and eosinophilic inflammation in mice. We hypothesized that acute SDS exposure would compromise the esophageal mucosal barrier in humans.

Methods:

Healthy adults brushed with 2 g of an SDS-containing toothpaste. Esophageal impedance was measured continuously pre-/post-toothbrushing to assess the effect of toothpaste on epithelial barrier function. SDS was measured in saliva using a methylene blue anionic substances assay. Participants completed 1-hr esophageal string tests (ESTs) pre-/post-toothbrushing and protein isolates from EST eluates from the proximal and distal esophagus were analyzed by tandem mass tag mass spectrometry.

Results:

Ten of twelve subjects completed the clinical study. SDS in the initial expectorate measured immediately after toothbrushing varied markedly (median: 321.40; range: 169.90–1,243.00 μg/mL) and was detectable in saliva in 80% of subjects (median: 9.55 μg/mL; range 0–123.40 μg/mL) at 60 min post toothbrushing. Esophageal mucosal impedance decreased within 30 min of toothbrushing (p<0.01). Toothbrushing increased salivary viscosity and proteins associated with mucosal defense in EST eluates. The tight junction pathway was downregulated while the salivary secretion and complement and coagulation cascades pathways were upregulated in the proximal esophagus.

Conclusion:

In healthy individuals, SDS remained detectable in saliva for a prolonged period after toothbrushing at concentrations previously shown to disrupt esophageal epithelial barrier function in vitro. Toothbrushing with an SDS-containing toothpaste was associated with reduced esophageal mucosal impedance, altered salivary properties, and activated mucosal immune responses.

Introduction:

Eosinophilic esophagitis (EoE) is a chronic type 2 inflammatory disease associated with symptoms of esophageal dysfunction and tissue eosinophilia. EoE incidence and prevalence have increased dramatically since the initial case descriptions in the 1970’s^1,2. Mechanistic studies suggest EoE stems from disruption of the esophageal epithelial barrier and environmental factors have been implicated in disease pathogenesis^3–5. However, the exposures that drive epithelial barrier dysfunction in EoE are unknown.

Coincident with the rise in allergic disease, detergents have been rapidly incorporated into household products. The same properties that make these synthetic surfactants excellent soap substitutes can disrupt cellular membranes, compromise mucosal integrity, and provoke inflammatory responses⁶. Akdis et al⁷ advanced the epithelial barrier hypothesis⁸, demonstrating anionic surfactants, including sodium dodecyl benzene sulfonate (SDBS) and sodium dodecyl sulfonate (SDS), also known as sodium lauryl sulfate (SLS), degrade airway epithelial tight junctions, increase permeability, and promote the release of IL-33^9,10. Humans are estimated to ingest 0.044 to 0.944 mg·kg⁻¹·day⁻¹ of anionic surfactants, and the primary source of oral detergent exposure is through toothpaste (0.01–0.9 mg·kg⁻¹·day⁻¹)¹¹. Indeed, SDS is commonly used in toothpaste at concentrations up to 5% w/w¹², which may affect the integrity of the esophageal epithelium.

Esophageal mucosal impedance has emerged as a functional biomarker of epithelial barrier integrity. In patients with eosinophilic esophagitis (EoE), decreased mucosal impedance correlates with impaired epithelial integrity, including dilated intercellular spaces¹³. These changes are thought to facilitate antigen penetration and promote type 2 inflammatory responses characteristic of EoE.

We previously used in vitro and mouse models to demonstrate that exposure to SDS decreases esophageal barrier integrity, stimulates IL-33 production, and promotes epithelial hyperplasia and tissue eosinophilia, reminiscent of EoE¹⁴. We hypothesized that SDS-containing toothpaste induces esophageal barrier dysfunction in humans. Herein, we show that SDS persisted in saliva up to an hour after toothbrushing. Further, toothbrushing with an SDS-containing toothpaste was associated with decreased esophageal mucosal impedance, altered biophysical and biochemical properties of saliva, and activation of local immune responses.

Methods:

Study Population

Healthy adult subjects (age ≥ 18 years) were recruited from Mayo Clinic Arizona as part of a greater-than-minimal risk protocol (IRB: 22–003963, NCT05482256) and enrolled following informed consent. Because esophageal mucosal impedance may be altered in gastrointestinal disease^15,16, subjects with a history of esophageal disease including but not limited to EoE, esophageal dysmotility, gastroesophageal reflux disease, history of dysphagia, chronic vomiting, chronic abdominal pain, unintentional weight loss, celiac disease, inflammatory bowel disease, irritable bowel syndrome, esophageal varices, chronic aspiration, connective tissue disorder, known parasitic infection, or pregnancy were excluded. A gelatin capsule is a component of the EST; therefore, subjects with a history of gelatin allergy were also excluded.

Study Design

We performed a case-crossover study where subjects were exposed to detergent-containing toothpaste by toothbrushing to evaluate its acute effects of exposure on saliva and the esophagus (Figure 1). Subjects were asked to avoid using toothpaste or mouthwash containing SDS, for at least two weeks before enrollment. On the morning of the study, subjects abstained from food and drink (except water) and refrained from brushing their teeth. The subject’s height was measured and recorded, and an EST was performed as previously described¹⁷. The EST is a gelatin capsule that contains a highly absorbent nylon string. The end of the string is taped to the cheek, and the capsule is swallowed with up to eight ounces of water. After one hour, the EST was removed and processed for laboratory analysis. After EST completion, a baseline saliva sample was collected by direct saliva expectoration into a 50 mL conical vial.

Figure 1. Study Design.

Subjects underwent an initial 1-hour esophageal string test (EST #1) and then high-resolution impedance manometry (HRIM). Baseline mucosal impedance was assessed for 10 minutes (min) and then the subject brushed for 2 min with 2 g of an SDS-containing toothpaste and expectorated without rinsing. Mucosal impedance was monitored for an additional 60 min. Finally, EST #2 was performed. Saliva was collected before HRIM, immediately post-toothbrushing (t = 0), 5 min post-toothbrushing (t = 5) and 60 min (t = 60) post-toothbrushing.

Decreased esophageal mucosal impedance is a marker of impaired mucosal barrier integrity^18,19. For esophageal impedance measurement, a topical nasal anesthetic (viscous lidocaine) was then administered, and a transnasal, solid-state esophageal high-resolution impedance manometry (HRIM) catheter was placed. Baseline mucosal impedance was assessed for ten minutes (min). The subjects then brushed their teeth and tongue using 2 g of commercial SDS-containing toothpaste¹² [SLS 1.5% (w/w)] for two min without swallowing with the catheter in place. Immediately after toothbrushing, subjects were instructed to fully expectorate oral contents into a sterile collection tube and were not permitted to rinse their mouths after brushing²⁰. Subjects were then allowed to swallow at will. Saliva (~2 mL) was obtained five min later to assess oral detergent concentration after toothbrushing and expectorating. Mucosal impedance was recorded continuously for one hour and a final saliva sample (~2 mL) was collected. The catheter was removed, and subjects rinsed their mouths. A second EST was performed after HRIM was completed. After one hour, the second EST was removed and processed.

SDS measurement

SDS belongs to a class of anionic surfactants called linear alkylene sulfates (LAS) which are among the most common methylene blue active substances (MBAS)²¹. The MBAS assay has been commonly used for detection of detergent concentrations in wastewater and has been adapted for measuring anionic surfactants in human urine^21,22. We used a commercially available MBAS assay (Instrumental Detergents Test, R-9423, CHEMetrics) to measure SDS concentrations in the commercial toothpaste and saliva. SDS (Sigma-Aldrich, St. Louis, MO) was used as a standard for quantification.

Salivary mucus rheology

The viscoelastic properties of saliva samples were measured by particle tracking microrheology (PTMR) as previously described^23–26. Briefly, 1 μm diameter microspheres with COOH surface chemistry were added to saliva samples and rotated for 30 min. The thermally driven motion of the particles was tracked as a function of time and the viscoelastic properties of each sample, including heterogeneity, were calculated from the Stokes-Einstein relationship^27–29. Mucus concentration and composition were determined by measuring the molecular weight of mucin macromolecules present in saliva via a combination of size exclusion chromatography with multi-angle laser light scattering and refractometry as previously described^{24,27,28,30,31}. Briefly, samples were diluted 10-fold with light scattering buffer (normal saline + 10mM EDTA and 0.01% sodium azide) and then eluted through a CL2B column which allowed mucins to pass through in the void fraction while smaller globular proteins were eluted in the included fraction. Molecular weight was calculated using a Berry Model to fit multi-angle laser light scattering data, while the concentration of the mucins was determined by refractometry.

Esophageal string test

The EST is a non-invasive, FDA-registered sampling device designed to collect biofluid from the esophagus.¹⁷ The patient’s height was used to determine the location of the upper and lower esophageal sphincters on the EST^32,33. The length of the esophageal segment was determined using a measuring tape and the esophageal portions of the string were cut into two segments: proximal and distal. These segments were snap frozen in liquid nitrogen and the proximal segment was used for proteomic analysis.

Mucosal impedance

To assess mucosal impedance, HRIM was performed using a solid-state esophageal catheter with 36 channels of circumferential pressure sensors and 18 impedance channels (4.2 mm). Mucosal impedance of the proximal and distal esophagus (mean of 4 sensors each) was extracted using ManoView^™ ESO v3.0.1 acquisition and analysis software. Sensors were selected relative to the upper and lower esophageal sphincters. Due to variability related to swallowing artifact, we applied a general additive model (GAM) with 20 basis functions to the generate impedance curves³⁴. We compared the median impedance at baseline (10 min period before brushing) to the nadir (lowest point of the curve within 30 minutes of brushing) of the impedance curves for the proximal and distal esophagus.

Proteomic analysis

Proteins were extracted from EST samples using 5% SDS. Total protein content was measured by bicinchoninic acid (BCA) assay. S-trap columns and protocol (Protifi.com) were used to adsorb the acidified proteins, remove SDS, and digest the samples with trypsin. The samples were grouped into 4 subsets that were tagged with TMT11-plex reagents. A bridge sample for normalizing tandem mass tag (TMT) signals among the four TMT11 sets was prepared by pooling equal total peptide amounts from each of the 40 samples. Equal aliquots of the bridge sample were included as the first reporter ion channel in each TMT11 set. Each TMT11 set was fractionated using basic pH reversed phase liquid chromatography (bRPLC) to 96 fractions that were recombined in a staggered pattern to 12 fractions to increase the depth of the proteome measured. Each of the 12 fractions was run by LC-MS3 (MS/MS/MS) on an Orbitrap Eclipse using a 2 hr LC gradient and real-time identification of peptides from MS2, followed by MS3 to measure reporter ion signal without interference from co-eluting peptides. Mass spec data was processed by MaxQuant to identify peptides from each fraction and sum the peptide MS3 abundances to their respective protein abundance. Protein reporter intensities were normalized by total sample signal (scaled to 1,000,000), then normalized within each TMT batch to the bridge reference channel, followed by batch correction using ComBat.

Statistical analysis

Descriptive statistics were used to summarize the study population. Repeated measures outcomes (salivary SDS concentration, percentage of solids, viscosity, and mucin concentration) were analyzed using repeated-measures one-way ANOVA or mixed-effects models with Geisser–Greenhouse correction, followed by Tukey’s multiple comparisons test. SDS concentration and viscosity were log-transformed prior to analysis. Paired comparisons of mucosal impedance were performed using the Wilcoxon signed-rank test. Correlations were assessed using Pearson’s coefficient. Proteomic data were analyzed using paired tests with false discovery rate (FDR) correction. Two-tailed p values <0.05 were considered statistically significant. Detailed statistical methods can be found in the supplementary material.

Manuscript preparation

The authors used ChatGPT (OpenAI, San Francisco, CA, USA) to assist with language editing and clarity. The authors reviewed and approved all content and take full responsibility for the final manuscript.

Results:

Ten of the 12 recruited, healthy subjects completed the study. Two subjects were unable to complete the HRIM study and were excluded from the analysis, one due to vomiting and the other due to difficulty with catheter placement because of small nares. Characteristics of the study participants are shown in Table 1. Consistent with prior experience, gagging occurred in approximately 1/3 of patients during EST placement³⁸. Among subjects that completed the study, none gagged during HRIM, and 2 (20%) gagged during toothbrushing with the HRIM catheter in place.

Table 1.

Characteristics of analyzed participants

Age in years (median, IQR)	31.50 (23.75–55.50)
Sex (n, %)
Female	3 (30.0%)
Male	7 (70.0%)
Race (n, %)
Other (%)*	5 (50.0%)
Not disclosed	1 (10.0%)
White	4 (40.0%)
Gagging (n, %)
EST placement	3 (30.0%)
HRIM catheter placement†	0 (0.0%)
Toothbrushing	2 (20.0%)

^*Other category includes African, Asian, and Mixed-race

^†Two subjects were unable to perform HRIM, toothbrushing, or the second EST

The study design is summarized in Figure 1. Briefly, ESTs were performed before and after HRIM. HRIM was measured continuously from t = −12 to t = 60 min after toothbrushing. Toothbrushing was performed from t = −2 to t = 0 min. Saliva was collected before HRIM (t= −15 min), immediately post-toothbrushing (t = 0 min), 5 min post-toothbrushing and 60 min post-toothbrushing.

To estimate the concentrations of SDS reaching the esophageal mucosa because of toothbrushing, we measured SDS in saliva at the timepoints shown in Figure 1. Trace levels of MBAS were detectable at baseline before toothbrushing (t= −15 min) [median (IQR) = 0.76 (16.76) μg/mL]. As expected, SDS concentrations were markedly increased after toothbrushing [mixed effects across timepoints F (2.21,19.86) = 56.16, p < 0.001] with median (IQR) = 321.4 (301.1) μg/mL at 0 min (Tukey’s test, p = <0.0001); (Figure 2A). Despite expectoration, SDS levels remained elevated at = 5 min [median (IQR) = 52.04 (66.94) μg/mL, Tukey’s test, p = 0.0002]. SDS was still detectable in the saliva of 80% of subjects at t = 60 min [median: 9.55 (44.08); range 0–123.40 μg/mL]. We noted considerable variability in SDS concentrations within the initial expectorate despite all subjects brushing with the same amount of toothpaste. This was likely due to a dilutional effect from individual variation in salivary production. Indeed, when salivary weight at t=0 was compared across subjects, those with the lowest SDS concentrations had the most salivary production (Figure 2B, Supplemental Figure 1). To understand the effect of SDS on salivary properties, we examined concentration (% solids), viscosity, and mucin concentration after toothbrushing. We noted at t = 0, there was a substantial amount of undissolved toothpaste; therefore, we excluded t = 0 data from our statistical analysis of overall saliva concentration, viscosity, and mucin concentration. We found toothbrushing generally increased salivary concentration (% solids) [F (1.691, 21.98) = 3.902, p = 0.04] (Figure 2C), viscosity [F (1.62, 14.61) = 4.36, p = 0.04] (Figure 2D), and mucin concentration [F (1.269, 9.520) = 12.88, p = 0.004] (Figure 2E) (Supplemental Table 1). We further note that the concentration of mucins present in saliva was significantly increased at t = 5 and t = 60 post-toothbrushing compared to the baseline measurements at t = −15 (Figure 2E, Tukey’s test, p = 0.005 for both). No changes in the molecular weight of mucins present in saliva were detected (data not shown) and saliva viscosity did not correlate with % solids or mucin concentration (Supplemental Figure 2).

Figure 2. Toothbrushing increased salivary SDS concentrations and altered the biophysical and biochemical properties of saliva.

(A) SDS concentrations in saliva were measured by a methylene anionic substances (MBAS) assay. Salivary SDS concentrations increased and remained elevated up 60 minutes later. (B) Salivary weight was assessed. Toothbrushing increased (C) salivary concentration (% solids), (D) viscosity, and (E) mucin concentration. Mean ± SEM shown for each, (n=10). Mixed effects analysis shown for each, * p < 0.05, ** p < 0.01

To investigate the acute effects of toothbrushing with an SDS-containing toothpaste on esophageal mucosal impedance as a surrogate of mucosal integrity, we performed continuous HRIM pre- and post-toothbrushing. Esophageal mucosal impedance decreased markedly after toothbrushing (Figure 3). Within 30 min, decreases in mucosal impedance were observed in the proximal or distal esophagus for all subjects [median change Ω (95%CI): proximal −840.5 (−1,319.0,−379.0); distal −690.0 (−951.3, −458.3), p < 0.01]. Absolute and relative change in impedance for each subject are reported in Supplemental Table 2. These decreases occurred immediately after toothbrushing and were sustained for up to 60 min (Figure 3, Supplemental Figure 3).

Figure 3. Esophageal mucosal impedance decreased markedly after toothbrushing.

(A) Geometrically smoothed, mucosal impedance curves before and after toothbrushing (hashed line) in the proximal and distal esophagus. Comparison of median mucosal impedance values at baseline vs. nadir in the (B) proximal and (C) distal esophagus. ** p < 0.01, Wilcoxon-signed-rank test, (n=10).

To assess the effects of toothbrushing with an SDS-containing toothpaste on the esophageal proteome, we compared EST-collected mucosal biofluids before and after toothbrushing and HRIM. Exploratory analysis by PCA suggested a modest treatment effect with heterogeneity within groups (Figure 4A). PC1 and PC2 accounted for 36.5% and 10.4% of the variance, respectively. Differences in post-treatment samples were driven by upregulation of proteins including cathepsin L1 (CTSL), cystatin D (CST5), and alpha-amylase 1A (AMY1A) and downregulation of heterogeneous nuclear ribonucleoprotein D-like (HNRNPDL), hydroxyacyl-CoA dehydrogenase (HADH), and single-stranded DNA-binding protein 1 (SSBP1). Differential expression analysis of paired samples revealed upregulation of twenty proteins and downregulation of seven proteins (Figure 4B). Specifically, we noted increases in mucosal defense and antimicrobial proteins (e.g., multiple cystatins, histatin-1, ribonuclease 4), secreted salivary proteins (e.g., lactoperoxidase, alpha-amylase 1A, basic salivary proline-rich protein 3), growth factors/signaling molecules (pleiotrophin, gremlin-2, CXCL17), and the lysosomal cysteine protease, cathepsin L1 (Supplemental Table 3). GSEA demonstrated downregulation of KEGG pathways associated with translation and RNA processing (ribosome, proteasome, and spliceosome pathways) and barrier function (tight junction pathway). Upregulated genes corresponded to activation of innate immune defense (complement and coagulation cascades pathway), mucosal immunity (salivary secretion, O-glycan biosynthesis pathways), and tissue remodeling (TGF-β signaling pathway) (Table 2). Regulation of key proteins in the complement and coagulation cascades, salivary secretion, and tight junction pathways are shown in Supplemental Figures 4–6.

Figure 4. The proteome of esophageal biofluid is altered with toothbrushing.

(A) Principal components analysis is shown with vector loadings for the top 16 proteins distinguishing pre- vs. post-treatment samples. (B) Volcano plot showing differentially expressed proteins in pre- vs. post-treatment samples. Proteins with log2FC ≥ 0.585 (fold change = 1.5) and −log10(FDR) = 1.259 (FDR = 0.1) are highlighted.

Table 2.

Results of pathway analysis for differential expression of proteins

Direction	GSEA analysis: post vs pre-toothbrushing pathways	NES	Proteins	adj. Pval
Down	Spinocerebellar ataxia	−0.507	41	3.5e-02
	Ribosome	−0.4385	62	7.1e-02
	Legionellosis	−0.6652	13	7.6e-02
	Proteasome	−0.4993	32	1.3e-01
	Tight junction	−0.4809	34	1.4e-01
	Tryptophan metabolism	−0.5995	14	1.5e-01
	Spliceosome	−0.43	44	1.5e-01
	Longevity regulating pathway-multiple species	−0.696	8	1.8e-01
	RNA degradation	−0.6422	10	1.8e-01
	Valine leucine and isoleucine degradation	−0.5296	19	1.8e-01
	Fatty acid degradation	−0.5293	19	1.8e-01
Up	Complement and coagulation cascades	0.8093	33	1.9e-09
	Salivary secretion	0.807	21	1.5e-05
	Cholesterol metabolism	0.6766	17	7.6e-02
	Fat digestion and absorption	0.7771	8	1.3e-01
	TGF-beta signaling pathway	0.7888	7	1.4e-01
	Mucin type O-glycan biosynthesis	0.7342	9	1.4e-01
	Other types of O-glycan biosynthesis	0.7093	9	1.6e-01
	Protein digestion and absorption	0.7868	6	1.8e-01
	ECM-receptor interaction	0.6323	13	1.8e-01

Discussion:

The environmental factors driving epithelial barrier dysfunction in EoE are unknown. Identification of these factors may explain the dramatic rise in EoE prevalence. We and others have previously shown that detergents disrupt epithelial barriers in the skin, lung, and esophagus^9,10,14,39. SDS is a common household detergent, and our primary oral exposure occurs likely through toothpaste¹¹. We previously found SDS concentrations at 0.5% w/v, approximately 1/6^th the concentration found commonly in toothpaste, induced marked EoE-like pathology in mice.¹⁴ Therefore, we hypothesized that brushing with an SDS-containing toothpaste would compromise esophageal mucosal integrity in humans.

To test this hypothesis, we used several novel approaches to assess the effects of SDS-containing toothpaste on the esophageal barrier including assessment of salivary SDS concentration through an MBAS assay, particle tracking microrheology, continuous esophageal impedance monitoring, and proteomic analysis of esophageal biofluid. Previous in vitro experiments using esophageal organoid culture, submerged culture, and air-liquid interface culture, showed that very low concentrations of SDS (≤ 5 μg/mL) disrupted epithelial structure, stimulated IL-33 production, and induced epithelial barrier dysfunction.¹⁴ In this study, we found that toothbrushing increased salivary SDS levels that persisted at pathologically relevant concentrations; indeed, median SDS concentrations of 9.55 μg/mL were detected in saliva one hour after toothbrushing. This was associated with acute decreases in esophageal mucosal impedance and downregulation of proteins in the tight junction pathway. Importantly, decreased mucosal impedance is a well-described feature of EoE and is thought to reflect epithelial barrier disruption; however, the relevance of this finding in the current healthy cohort following acute exposure is uncertain. In parallel, salivary secretion and innate mucosal defense responses were activated. Finally, we noted marked changes in the concentration, viscosity, and mucin content of saliva following toothbrushing. A complementary study by Ardicli et al. (under consideration) demonstrates that SDS directly disrupts esophageal epithelial barrier function in organoid models, supporting the mechanistic basis of our observations. Taken together, these results suggest that toothbrushing with an SDS-containing toothpaste may be associated with impaired esophageal barrier function, activated immunologic pathways, and altered biophysical and biochemical properties of esophageal mucus.

Despite widespread exposure to detergents such as SDS, only a subset of individuals develops EoE. This suggests that additional factors, including genetic susceptibility, atopic predisposition, epithelial barrier resilience, and environmental co-exposures, likely modulate disease risk. Recent studies highlight the importance of gene–environment interactions and epithelial barrier integrity in determining susceptibility to EoE^40,41. Our findings may therefore be most relevant to susceptible individuals rather than the general population.

Our results highlight the importance of salivation as a mucosal defense response. We noted marked changes in salivary volume following toothpaste exposure. Salivary SDS concentrations varied according to the weight of the initial expectorate, suggesting salivary flow rates influence the concentration of SDS in the esophagus. We speculate that hyposalivation may increase the concentration of, and prolong exposure to, barrier disruptive agents and antigens. Indeed, salivation is considered a protective response to esophageal injury^42,43 and the impulse to imbibe may be a behavioral adaptation that augments this natural response⁴⁴.

We initially hypothesized that SDS-containing toothpaste would decrease salivary viscosity by interfering with hydrophobic interactions between mucins^45,46. Unexpectedly, we observed increased salivary viscosity independent of the percentage of solids introduced by toothbrushing. We observed increases in mucin concentrations and upregulation of pathways associated with glycan biosynthesis. Furthermore, salivary enzymes and basic salivary proline-rich proteins may complex with mucins to increase esophageal mucus viscosity⁴⁷. This, in turn, may prolong antigen contact with a disrupted esophageal epithelium. Unlike our previous work, salivary viscosity did not correlate with mucin concentration or molecular weight^26,48. Whether this indicates that toothbrushing is altering interactions between mucins will be a focus of future investigations.

We acknowledge several limitations. First, our study design does not allow attribution of the observed changes specifically to SDS. Because all participants brushed with an SDS-containing toothpaste without a non-SDS comparator, the findings may reflect effects of toothbrushing, mechanical stimulation, salivary flow, swallowing behavior, or other toothpaste constituents, and therefore represent associations rather than isolated effects of SDS. Notwithstanding, SDS was the only detergent or surfactant identified in the list of toothpaste ingredients¹². Second, baseline MBAS levels > 10 μg/mL were detected in 30% of subjects despite instructions to avoid SDS-containing products, possibly reflecting exposure to alternative surfactants or other MBAS-active compounds (e.g., bile acids)⁴⁹. Third, mucosal impedance may be affected by the charge of anionic compounds by increasing conductivity independent of tissue damage^50,51. Because impedance was measured during and after exposure to saliva containing toothpaste constituents, reductions may reflect changes in luminal chemistry rather than true alterations in epithelial barrier integrity, although persistently low impedance readings at one hour despite the absence of MBAS in some subjects argues against this as the sole explanation. Fourth, interpretation of the EST proteomic findings is limited by the temporal structure of the study, as the post-toothbrushing EST followed HRIM, prolonged intraluminal instrumentation, toothbrushing, repeated saliva sampling, and time. As such, the observed proteomic changes cannot be attributed specifically to SDS exposure and may instead reflect a composite response to mechanical stimulation, procedural effects, salivary alterations, or time-dependent physiological changes. Therefore, the proteomic findings should be interpreted as reflecting the overall experimental condition rather than an isolated effect of SDS. Finally, this study was conducted in healthy adults following a single, acute exposure without symptom assessment or histology. Therefore, the clinical relevance of these findings to EoE pathogenesis, disease activity, or prevention remains uncertain.

In the future, a randomized controlled study design with SDS and non-SDS containing toothpaste is needed to isolate the effects of SDS on the esophagus. Furthermore, this study only examined the effects of acute toothpaste exposure. Long-term, daily SDS exposure through toothbrushing may have potential cumulative effects on esophageal barrier function and remodeling. Our observation that SDS remained persistently elevated for one hour after expectoration has important implications for vulnerable populations including young children and individuals with developmental delays, who may swallow large quantities of SDS in toothpaste⁵². This may account, in part, for observed associations between EoE and autism spectrum disorder⁵³. Our results highlight the importance of salivation as a protective response to prevent esophageal injury. Controlled observational studies are needed to determine whether salivary flow rates at baseline vs. in response to noxious or antigenic stimuli confer increased risk for EoE. Our study focused on the effects of SDS in healthy subjects. Further studies are needed to evaluate the impact of SDS avoidance on individuals with EoE or those at risk. Additional research is needed to identify other environmental and genetic factors with deleterious or compounding effects on esophageal barrier function. Finally, the use of SDS-containing toothpaste as a vehicle for delivery of peanut oral immunotherapy may have unintended negative consequences⁵⁴.

In conclusion, we showed that toothbrushing with an SDS-containing toothpaste results in sustained increases in salivary SDS concentrations. These concentrations were associated with decreased esophageal mucosal impedance suggesting compromised mucosal integrity. Decreased mucosal impedance is a feature of EoE and is associated with dilated intercellular spaces on esophageal mucosal biopsies, which facilitate penetration of food antigens through the esophageal mucosa^18,55. Toothbrushing also increased salivary viscosity, mucin concentration, and esophageal biofluid content, potentially contributing to delayed esophageal clearance. Disruption of mucosal integrity by SDS may facilitate antigen penetration into the submucosal space while simultaneously inducing epithelial alarmin release (e.g., IL-33), resulting in adaptive type 2 immune responses¹⁴. Collectively, these observations extend our preclinical studies in vitro and in mice¹⁴ and suggest that detergents may influence esophageal biology in ways that could be relevant to EoE pathogenesis. Our findings suggest that common environmental exposures, such as detergent-containing toothpaste, may represent modifiable factors with potential relevance to treatment and prevention strategies in EoE.

Abbreviations Used:

ALI

air liquid interface

BCA

bicinchoninic acid

bRPLC

basic pH reversed phase liquid chromatography

DEP

differential enrichment analysis of proteomics data

EoE

eosinophilic esophagitis

eos/hpf

eosinophils per high power field

EST

esophageal string tests

FDR

false discovery rate

GAM

general additive model

GSEA

gene set enrichment analysis

HRIM

high-resolution impedance manometry

IQR

interquartile range

LAS

linear alkylene sulfates

LIMMA

linear models for microarray data

MBAS

methylene blue active substances

min

minutes

PCA

principal components analysis

PTMR

particle tracking microrheology

TMT

tandem mass tag

SDBS

sodium dodecyl benzene sulfonate

SDS

sodium dodecyl sulfate

SEM

standard error of the mean

SLS

sodium lauryl sulfate