And Oh No!...
Update on this previous story here...
Introduction from the pdf report right here. It was available by following the link in one of the links they considered to have verification of this data.
Environmental factors play an important role in the pathogenesis of various inflammatory and autoimmune diseases1. Inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are serious chronic inflammatory diseases of the gastrointestinal (GI) tract; they affect millions of people worldwide2. Genetic susceptibility, dysregulated immune response toward perturbed gut microbiota and environmental factors have been shown to contribute to the etiopathogenesis of IBD3. Although significant progress has been made to identify susceptible genes and understand the role of the immune system and the gut microbiota, similar advances in defining environmental risk factors for IBD have fallen behind.
There is a growing body of evidence that diet plays a pivotal role in the development of IBD4,5. IBD incidence is rising rapidly in developed countries, such as the United States and Canada, and developing countries with a dramatic “westernization” of lifestyle2. A western diet is characterized by high intakes of food additives, fats, red meats, and sugar, and low intake of fibers, triggering chronic intestinal inflammation6,7,8. Food additives, such as emulsifiers, stabilizers, and synthetic colorants are widely used to improve the texture, preservation, and aesthetics of processed food. Several studies have reported that high levels of these additives, such as maltodextrin9 and titanium dioxide10, as well as ingredients added during food processing including dietary emulsifiers (polysorbate-80 and carboxy-methylcellulose11) and artificial sweeteners12 alter the gut microbiome, increase intestinal permeability, decrease mucus barrier thickness, and promote colitis.
The use of synthetic colorants in dietary products has significantly increased over the past 50 years13. These compounds are metabolized to generate free aromatic amines in the gut lumen, some of which are potentially carcinogenic and mutagenic14,15. Among many azo dyes, Allura Red AC (FD&C Red 40 or E129) (AR) is the most widely used colorant in many countries 16 and can be found in commonly consumed dietary products aimed at children (e.g., breakfast cereals, beverages, and confectioneries). AR is a non-genotoxic sulfonated mono-azo red dye that is metabolized by intestinal bacteria through azo reduction 17. AR exerts pro-inflammatory properties by promoting oxidative stress through reactive oxygen species (ROS) generation and cyclooxygenase-2 (COX-2) expression in the rat liver and kidney 18. Moreover, 10 mg kg−1 of body weight AR administered orally resulted in significant DNA damage in the mouse colon 19. Despite being highly prevalent in our diet, it is poorly understood how AR influences intestinal inflammation.
Serotonin or 5-hydroxytryptamine (5-HT) is a neurotransmitter and a hormone that regulates GI physiological functions in response to environmental stimuli in the gut. Enterochromaffin (EC) cells are responsible for synthesizing the majority of our body’s 5-HT via the rate-limiting enzyme, tryptophan hydroxylase (TPH) 1; while enteric neurons contribute in small quantities via TPH2 20. Along with previous findings that 5-HT content and EC cell number were elevated in IBD patients 21,22,23, we have previously reported mice with lower 5-HT in the gut as a result of TPH1 knockout (Tph1−/−) showed reduced colitis severity after dextran sulfate sodium (DSS) 24. We also found that higher mucosal 5-HT resulted in pro-colitogenic gut microbiota, enhancing susceptibility to colitis 25. Recently, it was shown Turicibacter sanguinis expresses a neurotransmitter sodium symporter-related protein with sequence and structural homology to mammalian serotonin re-uptake transporter (SERT) 26, further supporting a role for 5-HT in shaping the gut microbial composition.
Though some of the azo dyes enhance 5-HT levels in the hypothalamus and brain stem 27 and modulate 5-HT synthesis in vitro 28,29, whether there is any interplay among AR, 5-HT and the gut microbiota in influencing colitis susceptibility is unknown. By utilizing experimental models of colitis, intestinal organoids and human cell culture system, we aim to unravel the role of AR in modulating intestinal 5-HT signaling and the gut microbiota in relation to the susceptibility of colitis.
In this work, through a screening of several common synthetic colorants in a model of human EC cells (BON cells), we discover that AR promotes 5-HT secretion. Using mouse models of acute and chronic colitis, we identify chronic, but not the intermittent, exposure for 12 weeks to AR enhances the susceptibility to colitis and that, colonic 5-HT is a key mediator. Higher colonic 5-HT levels and disruption of the epithelial barrier function via myosin light chain kinase (MLCK) by AR exposure are associated with perturbation of the gut microbiota in specific pathogen-free (SPF) mice, while AR exposure also induces mild colitis and elevates colonic 5-HT in naïve germ-free (GF) mice. The ability of AR exposure to induce colitis depends on the bio-availability of colonic 5-HT as evidenced by using mice lacking TPH1 or SERT. Although it remains elusive whether similar effects are observed in humans, our data indicate that chronic exposure to a common synthetic colorant AR promotes experimental colitis via colonic 5-HT in gut microbiota-dependent and independent pathway in mice.
Results
Allura Red AC exacerbates DSS-induced colitis in naïve C57BL/6 mice As our previous studies revealed a key role of 5-HT in increasing colitis susceptibility 24,25, we investigated the influence of several common food colorants on the production of 5-HT in BON cells (human EC cell model). Based on their widespread presence in food due to food processing, we selected AR, Brilliant Blue FCF (BB), Sunset Yellow FCF (SY), and Tartrazine Yellow (TY) 30, and treated BON cells for 24 h with these colorants. All colorants promoted 5-HT secretion and TPH1 mRNA levels (Supplementary Fig. 1), where AR showed the most pronounced effect at the lowest 1 pmol L−1. Based on these findings, we further explored the role of AR as a potential dietary factor in the pathogenesis of colitis.
To understand the effect of AR on colitis development, C57BL/6 mice were either fed a normal chow diet or exposed to AR via diet (100 ppm; a custom diet; TD.190960) for 12 weeks, followed by 3.5% DSS for 7 days. Mice were exposed to AR during DSS while control groups received the control diet (Fig. 1a). AR level was calculated based on the acceptable daily intake (ADI) in humans (7 mg kg−1 per body weight) using the previously described formula as a benchmark 12. Food intake between the groups was not different prior to DSS (Supplementary Fig. 2). Mice exposed to AR without DSS showed reduced body weight, which was further exacerbated in DSS (Fig. 1b). Compared with DSS-treated mice, AR-exposed DSS-treated mice (AR-DSS) showed an increased disease activity index (DAI) (Fig. 1c), which was associated with higher macroscopic scores (Fig. 1d), reduction in colonic lengths (Fig. 1e, f) and increased colonic weights (Fig. 1g). Fecal lipocalin-2 (LCN2) levels were higher in AR-DSS compared to their DSS counterparts (Fig. 1h). Histological scores (Fig. 1i, j) and colonic MPO levels (Fig. 1k) were also higher in AR-DSS compared to DSS-treated mice. In addition, colonic interleukin (IL)−1β, IL-6, and tumor necrosis factor (TNF)-α, were higher in AR-DSS (Fig. 1l) than their DSS counterparts, while the genes that regulate intestinal epithelial barrier function (zonula occludin-1 [ZO-1; Tjp1], and occludin [Ocln]), were reduced in AR-DSS compared to their DSS counterparts (Fig. 1m). A significant decrease in Muc2 mRNA levels was observed in mice exposed to AR without DSS compared to untreated mice (Fig. 1m), suggesting a compromised mucus layer prior to DSS. Furthermore, colonic 5-HT levels were elevated in mice exposed to AR without DSS compared to untreated mice, and on day 7 post DSS compared to their DSS counterparts (Fig. 1n). We also investigated the effect of AR via water in C57BL/6 mice and observed similar results on day 7 post DSS (Supplementary Figs. 3 and 4). These data indicate that AR exacerbates DSS-induced colitis in C57BL/6 mice.
Allura Red AC triggers an early development of colitis in CD4+CD45RBhi T cell-induced colitis model
To further probe the role of AR in the development of colitis, we used a well-established T cell transfer chronic colitis model. FACS-sorted wild-type (WT) CD4+CD45RBhi T cells (Supplementary Fig. 5) were intraperitoneally transferred into Rag1−/− mice, and at the time of reconstitution, mice were either fed normal chow diet or exposed to AR via diet for 5 weeks (Fig. 2a). Transfer of T cells induced increased body weight loss (Fig. 2b), increased DAI (Fig. 2c), higher macroscopic score (Fig. 2d), and reduced colonic lengths (Fig. 2e, f) in CD45RBhi-AR mice compared to CD45RBhi mice. In addition, fecal LCN2 levels (Fig. 2g) and histological score (Fig. 2h, i) were higher in CD45RBhi-AR mice than CD45RBhi mice. Furthermore, colonic IL-1β, IL-6, TNF-α, and interferon [IFN]-γ levels were also increased in CD45RBhi-AR mice compared to CD45RBhi mice (Fig. 2j). These findings indicate AR can promote colitis via CD4+ T cells in Rag1−/− mice.
Early life exposure to Allura Red AC enhances susceptibility to DSS-induced colitis
Exposure during early life influences the susceptibility for IBD development in later life 31. Four-week-old C57BL/6 mice were either fed normal chow diet or exposed to AR via diet for 4 weeks prior to induction of acute colitis with 3.5% DSS for 7 days. To examine whether early life exposure to AR primes mice to enhanced susceptibility to DSS-induced colitis, mice were not exposed to AR during DSS (Supplementary Fig. 7a). Mice exposed to AR showed increased body weight loss compared to their DSS counterparts on day 7 post DSS (Supplementary Fig. 7b). Compared with DSS counterparts, DSS-treated mice, which were exposed to AR, showed higher DAI (Supplementary Fig. 7c). This increased DAI was accompanied by higher macroscopic scores (Supplementary Fig. 7d) and reduction in colonic length (Supplementary Fig. 7e, f). Histological scores (Supplementary Fig. 7g, h) and colonic MPO levels (Supplementary Fig. 7i) were also higher in mice exposed to AR compared to their DSS counterparts on day 7 post DSS. In addition, colonic 5-HT levels were elevated in mice exposed to AR without DSS compared to untreated mice, and this increase was further substantiated on day 7 post DSS (Supplementary Fig. 7j). Moreover, AR potently increased colonic IL-1β, IL-6, and TNF-α levels in mice on day 7 post DSS compared to their DSS counterparts (Supplementary Fig. 7k). These data indicate early life exposure to AR primes mice to heightened susceptibility to DSS-induced colitis.
Allura Red AC induces low-grade colonic inflammation in naïve C57BL/6 mice
Body weight (Fig. 1b) and Muc2 mRNA levels were reduced (Fig. 1m), while colonic 5-HT levels were increased (Fig. 1n), in mice exposed to AR without DSS. These observations led us to examine whether AR promotes low-grade colonic inflammation without DSS (Fig. 3a). Mice were exposed to AR for 14 weeks (7 days more than the exposure in Fig. 1), and food intake was not different between the groups (Supplementary Fig. 8). Mice exposed to AR showed 4.41 ± 1.96 ng mL−1 of AR concentration in the serum and higher fecal LCN2 levels than control (Fig. 3b). These mice also showed higher macroscopic (Fig. 3c) and histological scores (Fig. 3d, e), and colonic MPO levels (Fig. 3f) than control mice. The number of 5-HT+ cells was also increased in the colon of AR-exposed mice (Fig. 3g, h), along with increased colonic 5-HT levels (Fig. 3i) and colonic IL-1β, IL-6, and TNF-α levels (Fig. 3j) than control mice. Moreover, genes related to antimicrobial responses, such as peroxisome proliferator-activated receptor gamma (Pparg), β-defensin 3 (Defb3), IL22 (Il22), and regenerating islet-derived protein (REG) 3 gamma (Reg3g), were down-regulated in the colon of mice exposed to AR compared to control mice (Fig. 3k).
Basically my take on it is a very direct warning that they're admitting to messing up, so the chances of them believing that it is irreversible at this point would be the reason of us being able to be privy (Have Access To) this information at this part of the game. If they showed "Hot Cheetos and Takis" two generations would vanish instantly from either not wanting to, or couldn't deal with that executive decision for their own health seems to be the way that it is written?
Help me out here... Based on what you have read so far on this one subject, what are your thoughts on ..;
What do you think that RED40 companies are going to "Repair" an immediately needed to get handled situation, and not to be Class-Action Lawsuited your way out of this one, and any other business caught choosing "Profit Over People Evil-ness?"
What are you personally going to do knowing what you know now about this subject?
Your Household?
Your Tribe?
Your Village?
Your People?
Your Planet?
Your Future?
Our Future?
Share if you care, share if you don't... Someone cares enough for the both of us!
Oh no!!! This is alarming! Thanks for educating us!