Chronic exposure to synthetic food colorant Allura Red AC promotes susceptibility to experimental colitis via intestinal serotonin in mice

Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract influenced by genetic susceptibility, immune dysregulation, gut microbiota, and environmental factors. Dietary patterns, particularly Western diets rich in additives such as emulsifiers, stabilizers, artificial sweeteners, and synthetic colorants, have been implicated in altering the gut microbiome, increasing intestinal permeability, and promoting colitis. Allura Red AC (AR; FD&C Red 40; E129) is one of the most widely used azo synthetic colorants and is prevalent in foods commonly consumed by children. AR is metabolized by intestinal bacteria and has been reported to promote oxidative stress and induce DNA damage in rodent tissues. Serotonin (5-HT), predominantly synthesized by enterochromaffin (EC) cells via TPH1 in the gut, is elevated in IBD and contributes to disease severity; mice lacking TPH1 show reduced susceptibility to colitis. Prior work also shows serotonin signaling shapes gut microbiota composition, suggesting a bidirectional relationship. The research question is whether chronic exposure to AR increases susceptibility to colitis and whether this effect is mediated through colonic 5-HT and alterations in the gut microbiota. The study aims to define how AR modulates intestinal 5-HT signaling, epithelial barrier function, and microbiota to influence colitis susceptibility.

Previous studies demonstrate that Western dietary components, including emulsifiers (e.g., polysorbate-80, carboxymethylcellulose), maltodextrin, titanium dioxide, and artificial sweeteners, can perturb gut microbiota, erode the mucus barrier, increase permeability, and exacerbate colitis. Synthetic azo dyes are increasingly used and can be metabolized by gut bacteria to aromatic amines, some with potential toxicity. AR has been shown to induce ROS and COX-2 in rat liver and kidney and to cause DNA damage in mouse colon at certain doses. Serotonin is elevated in IBD patients, with increased EC cells and reduced mucosal SERT expression; experimental models show 5-HT promotes intestinal inflammation, while Tph1 knockout reduces colitis severity. Gut microbes can interact with host serotonin; for example, Turicibacter sanguinis expresses a serotonin transporter-like protein that can influence colonization dynamics in response to 5-HT. Some azo dyes can modulate 5-HT synthesis or levels in the brain and in vitro, but interactions among AR, 5-HT, and microbiota in colitis have not been defined. Together, these works support investigating AR as a dietary factor affecting colitis via serotonergic and microbiome pathways.

- In vitro screening: BON cells (human EC cell model) were treated for 24 h with common food colorants (Allura Red AC, Brilliant Blue FCF, Sunset Yellow FCF, Tartrazine) to assess 5-HT secretion and TPH1 mRNA. AR effects on intracellular ROS (DCF-DA) and NF-κB involvement (triptolide pre-treatment) were evaluated. HT-29 colonic epithelial cells were used to examine AhR activation and CYP1A1/CYP1B1 mRNA induction; MLCK pathway activation assessed via Mlck mRNA and pMLCSer19, with or without TNF-α pre-treatment. - Murine models: C57BL/6 mice were exposed to AR via diet (100 ppm; TD.190960) chronically for 12 weeks prior to acute DSS colitis (3.5% DSS for 7 days), with continued AR during DSS. AR in drinking water (0.01% w/v) was also tested. An intermittent exposure regimen (1 day/week AR for 12 weeks) was evaluated. Early-life exposure: 4-week-old mice received AR for 4 weeks, followed by DSS without AR to test priming effects. - T cell transfer colitis: Rag1−/− mice received FACS-sorted WT CD4+CD45RBhigh T cells and were fed chow or AR-containing diet; disease progression was monitored for 5 weeks. - Genetic models: Tph1−/− mice were exposed to AR chronically and subjected to DSS to test 5-HT dependency. SERT-deficient mice were similarly evaluated for DSS severity with AR exposure. - Germ-free studies: GF mice received cecal microbiota transfer (CMT) from AR-exposed or control SPF donors (equal pools, 200 µL gavage daily ×3), then colonized for 21 days before 2.0% DSS for 7 days. Separately, naïve GF mice were chronically exposed to AR for 14 weeks without DSS to assess microbiota-independent effects. - Organoid models: Murine colonic crypt organoids were treated with TNF-α (10 ng/mL) followed by AR (1 µM) to evaluate morphological disruption. 2D monolayers derived from organoids were treated similarly to assess Mlck mRNA, ZO-1 localization, and barrier-related gene expression. - Outcomes: Body weight, disease activity index (DAI), macroscopic damage, colon length and weight, fecal lipocalin-2 (LCN2), myeloperoxidase (MPO), histology (H&E scoring), goblet cell counts (PAS staining), cytokines (IL-1β, IL-6, TNF-α, IFN-γ), epithelial tight junction markers (ZO-1 protein; Tjp1, Ocln mRNA), mucus gene (Muc2), antimicrobial/mucosal genes (Pparg, Defb3, Il22, Reg3g), MLCK pathway markers (Mlck mRNA, pMLCSer19), colonic 5-HT content and EC cell counts (IF for 5-HT), and serum AR levels (LC-MS/MS). - Microbiome profiling: 16S rRNA (v3–v4) sequencing of cecal contents with DADA2, SILVA taxonomy assignment, beta-diversity (Bray-Curtis), and differential relative abundance at phylum and genus levels. - Statistics: Two-tailed unpaired t-tests, one-way or two-way ANOVA with Bonferroni/Dunnett post hoc tests, or Mann–Whitney U as appropriate; P < 0.05 considered significant. - Dosing details: AR diet 100 ppm for 12–14 weeks; AR water 0.01% w/v; DSS 2.0% or 3.5% for 7 days depending on experiment. Sample sizes varied per experiment as indicated in figure legends.

- AR increases 5-HT secretion in BON cells and upregulates TPH1 mRNA, with detectable effects at very low concentrations (as low as 1 pmol/L for some dyes; AR most pronounced). AR elevates intracellular ROS and 5-HT release; NF-κB inhibition (triptolide) attenuates these effects. - Chronic AR exposure (diet, 100 ppm, 12 weeks) exacerbates DSS-induced colitis in C57BL/6 mice: greater weight loss, increased DAI, higher macroscopic and histological scores, shortened colons, increased colonic weight, elevated fecal LCN2, MPO, and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Colonic 5-HT levels are increased with AR both basally and post-DSS. - T cell transfer model: AR exposure accelerates and worsens CD4+CD45RBhigh-induced colitis in Rag1−/− mice, with higher DAI, macroscopic/histological scores, LCN2, and elevated IL-1β, IL-6, TNF-α, IFN-γ. - Intermittent AR exposure (1 day/week for 12 weeks) does not affect DSS colitis severity, indicating exposure pattern matters. - Early-life AR exposure (4 weeks in young mice) primes for heightened susceptibility to DSS colitis later, with increased DAI, macroscopic/histological severity, MPO, cytokines, and elevated colonic 5-HT. - AR alone induces low-grade colonic inflammation in naïve mice (14-week exposure): serum AR detectable (4.41 ± 1.96 ng/mL), increased LCN2, macroscopic/histological scores, MPO, elevated numbers of 5-HT+ EC cells and colonic 5-HT, increased IL-1β, IL-6, TNF-α, and downregulation of Pparg, Defb3, Il22, Reg3g. - Epithelial barrier disruption associated with MLCK activation: increased Mlck mRNA and pMLCSer19; decreased ZO-1 protein and Tjp1 mRNA; reduced PAS+ goblet cells and Muc2 expression. HT-29 cells and organoid monolayers show AR-enhanced MLCK signaling and barrier alterations, especially with TNF-α pre-treatment. - Mechanistic role of 5-HT: In Tph1−/− mice, AR exposure does not exacerbate DSS colitis, indicating dependence on mucosal 5-HT synthesis. In SERT-deficient mice (enhanced 5-HT bioactivity), AR exposure further increases DSS colitis severity and colonic 5-HT (as noted in discussion). - Microbiome changes: Chronic AR alters cecal microbiota composition (distinct Bray–Curtis clustering; shifts at phylum and genus levels, including changes in Turicibacter, Desulfovibrio, Roseburia, Lactobacillus, Muribaculum, Mucispirillum, Parabacteroides; Escherichia and Klebsiella not detected). Cecal microbiota transfer from AR-exposed donors to GF mice increases DSS colitis severity, MPO, histology scores, and elevates colonic 5-HT and cytokines compared to transfer from control donors. - Microbiota-independent effects: In GF mice, chronic AR exposure (14 weeks) increases MPO and histological scores and elevates colonic 5-HT and EC cell numbers even without microbial signals; cytokines were not significantly changed, indicating both microbiota-dependent and -independent pathways. - Overall, chronic, but not intermittent, AR exposure promotes colitis susceptibility via increased colonic 5-HT and MLCK-mediated epithelial barrier dysfunction, with contributions from altered gut microbiota.

The study demonstrates that chronic exposure to the synthetic food colorant Allura Red AC increases susceptibility to experimental colitis through mechanisms centered on colonic serotonin and epithelial barrier disruption. AR elevates mucosal 5-HT by directly stimulating EC cells via ROS generation and NF-κB activation, and by modulating the gut microbiota. Elevated 5-HT is linked to impaired barrier function, including MLCK activation and tight junction dysregulation, and decreased mucus (Muc2) and goblet cells, which together predispose to inflammation. The lack of AR effect in Tph1−/− mice underscores the necessity of TPH1-derived mucosal 5-HT for AR-induced colitogenic effects, while the exacerbation in SERT-deficient mice aligns with increased 5-HT bioactivity worsening inflammation. Microbiome analyses reveal AR-induced dysbiosis that, upon transfer to GF mice, confers heightened colitis and increased colonic 5-HT, demonstrating a microbiota-mediated component. However, AR also elevates colonic 5-HT and induces low-grade inflammation in GF mice, indicating microbiota-independent actions. The exposure pattern is critical: continuous long-term AR exposure increases risk, while intermittent exposure under the tested regimen did not. These findings connect a common dietary additive to disrupted intestinal homeostasis and highlight serotonergic signaling and MLCK-mediated barrier dysfunction as key mediators, with potential implications for populations with high colorant intake, particularly children.

Chronic long-term exposure to Allura Red AC, at a dose relevant to commonly consumed products, increases susceptibility to experimental colitis in mice by elevating colonic serotonin and impairing epithelial barrier function via MLCK. The effect requires TPH1-derived mucosal 5-HT and involves both microbiota-dependent and microbiota-independent pathways. Intermittent exposure over the tested period did not increase colitis susceptibility, whereas early-life exposure primes for heightened disease risk later. These results suggest that widespread use of AR could pose risks for intestinal inflammatory diseases and warrant further investigation in humans. Future work should: (1) assess dose–response relationships and exposure durations, including intermittent patterns over longer periods; (2) examine combined effects with other common dyes (e.g., Brilliant Blue, Sunset Yellow, Tartrazine); (3) delineate specific microbial taxa (e.g., Turicibacter) and mechanisms linking AR, 5-HT, and dysbiosis; and (4) conduct population-based studies to evaluate associations between AR intake and IBD risk and activity.

- Single AR dose was tested; dose–response effects and real-world exposure variability were not addressed. - The intermittent exposure regimen (1 day/week for 12 weeks) may have been insufficient in duration or intensity to reveal effects; longer or different intermittent schedules were not evaluated. - Translational relevance to humans remains unknown; human exposure patterns and metabolic differences may alter outcomes. - The microbially derived metabolite p-cresidinesulfonic acid (CS) was not assessed in vivo, though it did not stimulate 5-HT in vitro. - Potential saturation of bacterial azoreductase at high AR concentrations and consequent accumulation of parent compound was not directly tested. - While microbiome shifts were characterized at phylum/genus levels, causal taxa and mechanisms require further validation; synergy with other food additives/dyes was not explored.