The association between mycotoxin levels in urine of children and the risk of Crohn’s disease

The association between mycotoxin levels in urine of children and the risk of Crohn’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The association between mycotoxin levels in urine of children and the risk of Crohn’s disease Kai Zheng, Ning Shen, Zhongqin Jin, Jie Zhang, Jian Huang, Bin Xue, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6711624/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Crohn’s disease (CD), a chronic inflammatory bowel disease with increasing pediatric incidence, is influenced by environmental factors, including potential dietary contaminants like mycotoxins. This study investigated the association between urinary levels of two mycotoxins, zearalenone (ZEN) and aflatoxin B1 (AFB1), and CD risk in children. In a case-control study involving 32 children with CD and 30 healthy controls, urinary ZEN and AFB1 concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry. Urinary ZEN concentrations were significantly higher in CD cases compared to controls (median 0.33 pg/ml vs 0.16 pg/ml, P < 0.001), whereas no significant difference was observed for AFB1 levels (P = 0.989). Age-adjusted logistic regression analysis revealed a strong, dose-dependent positive association between higher urinary ZEN quartiles and CD risk; compared to the lowest 50%, adjusted Odds Ratios for the third and fourth quartiles of ZEN were 47.94 (P = 0.0001) and 62.93 (P = 0.0002), respectively. No significant association was found between urinary AFB1 levels and CD risk. Receiver Operating Characteristic analysis showed ZEN had superior discriminatory ability for CD (Area Under Curve [AUC] = 0.92) compared to AFB1 (AUC = 0.50). These findings demonstrate a strong positive association between elevated urinary ZEN levels and pediatric CD risk, suggesting ZEN exposure, potentially linked to its estrogenic properties, as an environmental risk factor. Conversely, urinary AFB1 was not associated with CD risk in this cohort, highlighting the potential role of specific mycotoxins in pediatric CD pathogenesis and warranting further investigation. Health sciences/Gastroenterology/Gastrointestinal diseases Earth and environmental sciences/Natural hazards Crohn’s disease Mycotoxin Zearalenone Aflatoxin B1 Figures Figure 1 Introduction Crohn’s disease (CD) is a chronic, relapsing inflammatory condition characterized by transmural inflammation that can affect any part of the gastrointestinal tract, from the mouth to the anus 1 . The global incidence and prevalence of CD have been steadily increasing, particularly in newly industrialized countries and notably among children and adolescents 2 , 3 . Pediatric-onset CD often presents with a more severe disease course and significantly impacts growth, pubertal development, bone health, quality of life, and psychosocial well-being 4 , 5 . While the exact etiology of CD remains incompletely understood, it is widely accepted to result from a complex interplay between genetic susceptibility, immune system dysregulation, and various environmental factors 1 , 6 . Environmental factors are gaining increasing attention in the pathogenesis of CD, as changes in environmental exposures are thought to underlie the rapid rise in incidence observed over recent decades, which cannot be solely explained by genetic shifts 6 , 7 . These factors encompass a wide range, including diet, smoking, antibiotic use, hygiene conditions, infections, and exposure to environmental contaminants 7 , 8 . Among dietary contaminants, mycotoxins which is the toxic secondary metabolites produced by fungi that commonly contaminate food staples represent a potential class of environmental triggers warranting investigation due to their ubiquitous nature and diverse biological effects 9 , 10 . This study focuses on two prevalent mycotoxins: Aflatoxin B1 (AFB1) and Zearalenone (ZEN). AFB1, primarily produced by Aspergillus flavus and Aspergillus parasiticus , is a frequent contaminant of cereals (especially maize), oilseeds, spices, and nuts 11 . It is classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC) due to its potent hepatotoxicity and carcinogenicity, mediated through DNA adduct formation following metabolic activation 11 , 12 . Beyond its carcinogenic potential, AFB1 has also been suggested to exert immunomodulatory effects, potentially altering immune cell function and cytokine production 13 . Zearalenone (ZEN), produced mainly by Fusarium species, is commonly found in maize, wheat, barley, and other grains 14 . ZEN is structurally similar to endogenous estrogens and exhibits potent estrogenic activity by binding to estrogen receptors, classifying it as a mycoestrogen or xenoestrogen 15 . This endocrine-disrupting property can affect the reproductive system, but ZEN has also been shown to modulate immune responses, potentially influencing inflammatory processes 16 , 17 . We chose to investigate AFB1 and ZEN because they are common dietary contaminants worldwide, and their respective toxicological profiles including potential immune modulation (AFB1, ZEN) and significant endocrine-disrupting activity (ZEN) suggest plausible mechanisms through which they might influence intestinal inflammation and CD risk. Despite the potential relevance, research specifically investigating the association between exposure to AFB1 and ZEN and the risk of CD, particularly in pediatric populations, remains limited and inconclusive 9 , 18 . While assessing exposure accurately is crucial, utilizing urinary biomarkers offers an objective measure of recent internal exposure, overcoming limitations associated with dietary recall methods 19 , 20 . Therefore, investigating the relationship between urinary levels of these specific mycotoxins and pediatric CD is necessary to clarify their potential role as environmental risk factors. Given this background, the primary objective of this case-control study was to investigate the association between urinary concentrations of AFB1 and ZEN and the risk of Crohn’s disease among Chinese children. We hypothesized that higher urinary levels of ZEN and/or AFB1 would be associated with an increased risk of developing CD in this population. A secondary objective was to evaluate the potential screening performance of urinary ZEN and AFB1 levels as biomarkers for discriminating between children with CD and healthy controls. Methods and Materials Participants This study was conducted at the forth affiliated hospital of Soochow university between September 2021 and December 2022. The study protocol was approved by the ethics committee of the forth affiliated hospital of Soochow university (Approval Number: 180005). Written informed consent was obtained from the parents or legal guardians of all participants, and assent was obtained from children capable of understanding the study’s purpose. Inclusion criteria for cases : Patients (aged 8 to 18 years) either newly diagnosed with CD (within 1 year) or those with established disease who had relapsed within 3 months according to Consensus Guidelines for the Diagnosis and Treatment of Inflammatory Bowel Disease 21 were eligible for inclusion as cases (n = 32). Exclusion criteria for cases : Patients with indeterminate colitis, co-existing significant gastrointestinal infections at the time of diagnosis, known genetic syndromes associated with IBD-like symptoms, or severe concomitant systemic diseases were excluded. Patients who had already started specific CD treatments like biologics or extensive immunosuppression before sample collection was also excluded. Inclusion criteria for controls : Healthy children (n = 30) visiting the hospital during the same period for routine health check-ups or minor, non-gastrointestinal, non-immunological conditions (e.g., minor trauma, scheduled vaccinations) were recruited as controls. Controls were selected to be generally comparable in age range to the cases. Exclusion criteria for controls : Children with any history of chronic gastrointestinal disease, inflammatory conditions, known immune deficiencies, malabsorption syndromes, chronic liver or kidney disease, or recent use of medications known to affect gut function or mycotoxin metabolism. Clinical Assessments Demographic information, including age and gender, was collected for all participants through standardized questionnaires administered to the parents/guardians and/or review of medical records. For CD cases, relevant clinical data pertaining to diagnosis confirmation were extracted from medical records. Sample Collection and Preparation Spot urine samples (first morning void) were collected from all participants using sterile, single-use containers. Immediately after collection, samples were centrifuged at 3000 rpm for 10 minutes at 4°C to remove cellular debris. The supernatant was carefully transferred into labeled polypropylene tubes, aliquoted, and stored frozen at -80°C until analysis. Analysis of Urinary Mycotoxins by HPLC-MS/MS Urinary concentrations of AFB1 and ZEN were quantified using a validated HPLC-MS/MS method 22 . Briefly, urine samples (10mL) underwent an enzymatic hydrolysis step using 10ul β-glucuronidase/arylsulfatase (≥ 100,000 units/mL, Sinopharm, Shanghai, China) to release conjugated forms of the mycotoxins. The mixture was then incubated at 25°C overnight in a water bath incubator (YIHENG, Shanghai, China) to complete the enzymatic hydrolysis. After enzymatic hydrolysis, the sample was extracted using an SPE column (C18 ENVI, 0.25 g). The extract was eluted with methanol (2mL). Subsequently, the solvent was evaporated to dryness using a Termovap sample concentrator (Baojing, Zhengzhou, Henan, China) operated within a biological safety cabinet. Drying was achieved under a stream of high-purity nitrogen gas (≥ 99.9%, Baoshan Praxair, Shanghai, China) delivered at a flow rate of 3L/min (outlet pressure set to 0.3 MPa). Finally, methanol (100 µL) was used to redissolve the analyte. Fifty microliters of the analyte solution was transferred using a 100µL microsyringe (Shimadzu, Kyoto, Japan) to a 1.5mL autosampler vial (Shimadzu, Kyoto, Japan) for subsequent injection and analysis of mycotoxin levels. Detection and quantification were performed using a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode, monitoring specific precursor-to-product ion transitions for AFB1 and ZEN. The 1 µg/mL standard working solutions of AFB1 and ZEN (≥ 99%, TargetMol, Wellesley Hills, MA, USA) were prepared with methanol (≥ 99.9%, Supelco, Bellefonte, PA, USA) as solvent. Quality control measures, including procedural blanks, spiked samples, and replicate analyses, were incorporated throughout the analytical process to ensure data reliability. Statistical Analyses All statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize participant characteristics. Differences in demographic characteristics and urinary mycotoxin levels between the case and control groups were assessed using the Mann-Whitney U test for continuous variables and the Pearson Chi-square (χ²) test. To evaluate the association between urinary mycotoxin concentrations and the risk of CD, binary logistic regression analysis was performed. Log-transformed concentrations of urinary AFB1 and ZEN were then categorized into quartiles based on the distribution in the total population. The lowest two quartiles (Q1 + Q2) were combined and used as the reference category. ORs and their corresponding 95% CIs were calculated for the third quartile (Q3) and fourth quartile (Q4) relative to the reference group (Q1 + Q2). The logistic regression models were adjusted for age as a potential confounder. ROC curve analysis was conducted to assess the discriminatory ability of urinary ZEN and AFB1 concentrations to distinguish between CD cases and healthy controls. The AUC with its 95% CI was calculated for each mycotoxin. A two-sided P-value < 0.05 was considered statistically significant for all analyses. Results Demographic characteristics of study participants A total of 62 children were enrolled in this study, comprising 32 children diagnosed with CD cases and 30 healthy children (controls). The demographic characteristics of the participants are presented in Table 1 . The median age of the CD cases (12 years, IQR: 11–14 years) was significantly younger than that of the control group (13 years, IQR: 13–14 years) (Z = -2.17, P = 0.030). There was no statistically significant difference in gender distribution between the case group (17 males, 53.10%) and the control group (15 males, 50.00%) (χ² = 0.06, P = 0.806). Table 1 The demographics for cases and control Control (n = 30) Case (n = 32) Z/χ2 P Value 25th Median 75th 25th Median 75th Age 13 13 14 11 12 14 -2.17 0.030 Male(%) 15(50.00) 17(53.10) 0.06 0.806 Aflatoxin B1 (pg/ml) 14.00 19.78 27.93 14.07 19.60 26.88 -0.01 0.989 Zearalenone(pg/ml) 0.15 0.16 0.21 0.26 0.33 0.36 -5.73 < 0.001* Comparison of urinary mycotoxin levels Urinary concentrations of AFB1 and ZEN were compared between CD cases and controls, as shown in Table 1 . No significant difference was observed in the median urinary AFB1 concentrations between the CD cases (19.60 pg/ml, IQR: 14.07–26.88 pg/ml) and the control group (19.78 pg/ml, IQR: 14.00-27.93 pg/ml) (Z = -0.01, P = 0.989). In contrast, urinary ZEN concentrations were significantly higher in children with CD compared to healthy controls. The median urinary ZEN level in the case group was 0.33 pg/ml (IQR: 0.26–0.36 pg/ml), which was substantially and statistically significantly higher than the median level of 0.16 pg/ml (IQR: 0.15–0.21 pg/ml) found in the control group (Z = -5.73, P < 0.001). Association between urinary mycotoxin concentrations and Crohn’s disease risk The associations between log-transformed urinary mycotoxin concentrations (categorized into quartiles, with Q1 + Q2 combined as the reference) and the risk of CD were evaluated using logistic regression analysis, with results presented in Table 2 . Table 2 Associations of mycotoxin concentrations with Crohn's disease (n = 62). Mycotoxins Control(%) Case(%) OR* 95% C.I. P Value Aflatoxin B1 (Log transformed) Q1 + Q2 15(50.00) 16(50.00) 1 Q3 7(23.30) 9(28.10) 1.12 0.31–3.98 0.8670 Q4 8(26.70) 7(21.90) 1.01 0.28–3.64 0.9940 Zearalenone (Log transformed) Q1 + Q2 25(83.30) 5(15.60) 1 Q3 3(10.00) 15(46.90) 47.94 6.54-351.77 0.0001* Q4 2(6.70) 12(37.50) 62.93 7.31-541.87 0.0002* *These ORs were adjusted for age and gender. For AFB1, after categorizing log-transformed concentrations, no significant association with CD risk was found. Compared to the reference group (Q1 + Q2, 50.00% cases vs. 50.00% controls), children in the third quartile (Q3) of AFB1 exposure had an Odds Ratio (OR) of 1.12 (95% Confidence Interval [CI]: 0.31–3.98, P = 0.8670). Similarly, children in the fourth quartile (Q4) of AFB1 exposure showed an OR of 1.01 (95% CI: 0.28–3.64, P = 0.9940) for CD risk. A strong, dose-dependent positive association was observed between higher urinary ZEN concentrations and the risk of CD. In the reference group (Q1 + Q2), 83.30% were controls and 15.60% were cases. For children in the third quartile (Q3) of log-transformed ZEN exposure, the risk of CD was markedly increased, with an OR of 47.94 (95% CI: 6.54-351.77, P = 0.0001) compared to the reference group. This association was even stronger for children in the highest quartile (Q4) of ZEN exposure, who had an OR of 62.93 (95% CI: 7.31-541.87, P = 0.0002) for developing CD. These ORs were all adjusted for age and gender. Screening performance of urinary mycotoxins for Crohn’s disease ROC curve analysis was performed to evaluate the ability of urinary ZEN and AFB1 concentrations to discriminate between children with CD and healthy controls. The results of this analysis are depicted in Fig. 1 . The AUC for ZEN was 0.92 (95% CI: 0.85 to 1.00), indicating good discriminatory power. In contrast, the AUC for AFB1 was substantially lower at 0.50 (95% CI: 0.35 to 0.65), suggesting poor discriminatory ability, not significantly different from chance. The ROC curve for ZEN was positioned notably higher and to the left compared to the curve for AFB1, visually confirming its superior performance in distinguishing between the two groups. Based on the ZEN ROC curve, an optimal cut-off value of 0.205 pg/mL yielded a sensitivity of 100% and a specificity of 76.7%. Discussion This study investigated the association between urinary levels of two common dietary mycotoxins, ZEN and AFB1, and the risk of Crohn’s disease in a pediatric cohort. The principal finding was a strong, significant, and dose-dependent positive association between elevated urinary ZEN concentrations and an increased risk of CD. In contrast, no significant association was observed between urinary AFB1 levels and CD risk. These results suggest that exposure to ZEN, but not necessarily all mycotoxins, may be an important environmental risk factor for pediatric CD, potentially linked to its estrogenic properties. The robust association between ZEN and pediatric CD warrants a detailed exploration of potential underlying biological mechanisms. ZEN is well-characterized as a potent mycoestrogen, structurally similar to endogenous estrogens, enabling it to bind to and activate estrogen receptors (ERs) 23 , 24 . This estrogenic activity is a plausible link to CD pathogenesis through several pathways. Firstly, ZEN can exert significant immunomodulatory effects 16 . Estrogens are known to influence the balance of T helper (Th) cell subsets, such as Th1, Th2, and Th17, and modulate the production of various cytokines involved in inflammatory processes 25 , 26 . ZEN, by mimicking estrogen, could dysregulate these critical immune responses within the gut. For instance, studies have shown ZEN can alter cytokine profiles and immune cell functions 16 , 27 . Secondly, ZEN exposure has been linked to the disruption of intestinal barrier integrity 28 . Increased intestinal permeability (“leaky gut”) is a key feature in CD pathogenesis, allowing increased translocation of luminal antigens and triggering inflammatory responses 29 . ZEN has been demonstrated to compromise intestinal epithelial tight junctions and increase permeability in in vitro and in vivo models 28 , 30 . Thirdly, the gut itself is an estrogen-responsive organ, expressing ERα, ERβ, and G protein-coupled estrogen receptor 1 (GPER1) 28 . ZEN, acting through these receptors on intestinal epithelial cells, immune cells, or even enteric neurons, could directly modulate local inflammatory pathways. Finally, emerging evidence suggests that estrogens and xenoestrogens can influence the composition and function of the gut microbiome, which plays a crucial role in IBD 31 . Alterations in the gut microbiota by ZEN could further contribute to intestinal dysbiosis and inflammation. These interconnected mechanisms provide a biologically plausible framework for ZEN’s involvement in CD. While AFB1 is a potent hepatotoxin and carcinogen 32 , 33 , and has been shown to possess some immunomodulatory properties 34 , its primary toxicological impact might be less directed towards the specific immune dysregulation seen in CD compared to the estrogenic effects of ZEN. The main target organ for AFB1 toxicity is the liver, and its effects on gut immunity might be secondary or less pronounced. Comparing our findings with existing literature, studies on mycotoxins and IBD, particularly pediatric CD, are limited. It was reported that food-associated mycotoxins, including ZEN, could be risk factors in individuals predisposed to chronic intestinal inflammatory diseases by affecting intestinal permeability and immune responses 9 . Studies investigating endocrine-disrupting chemicals (EDCs) have also reported associations with IBD or immune dysregulation 35 , 36 , lending indirect support to our findings regarding ZEN. Our finding of a strong association for ZEN aligns with experimental evidence showing its pro-inflammatory and barrier-disrupting effects in intestinal models. Our study adds specific evidence regarding ZEN and AFB1 in a pediatric CD context, highlighting ZEN as a particularly relevant exposure. In-depth mechanistic studies, utilizing animal models of IBD, intestinal organoids, and in vitro cell culture systems, are crucial to delineate the precise molecular pathways through which ZEN impacts intestinal inflammation, barrier function, immune cell responses, and the gut microbiome. Several limitations of this study should be considered. The case-control design inherently limits our ability to establish causality, and though biomarkers reduce recall bias, selection bias might still be present. The relatively small sample size (n = 62) resulted in wide confidence intervals for the odds ratios and reduced the statistical power for more granular analyses, impacting the precision of our estimates. While a single spot urine sample provides an objective measure of recent exposure, it may not fully capture long-term or intermittent exposure patterns crucial for chronic disease development. Other potential confounders, such as detailed dietary intake patterns, specific genetic susceptibilities, socioeconomic status, and co-exposure to other environmental agents, were not assessed and could influence the observed associations. Finally, the study was conducted in a specific geographical population, which may limit the generalizability of the findings to other populations with different dietary habits, genetic backgrounds, or mycotoxin exposure profiles. Consequently, larger-scale prospective studies are warranted to confirm these findings and further elucidate the role of mycotoxin exposure in pediatric CD. Conclusion This study provides compelling evidence for a strong, dose-dependent positive association between elevated urinary ZEN concentrations and an increased risk of Crohn’s disease in the pediatric population investigated. The observed discriminatory ability of urinary ZEN further supports its potential relevance as a biomarker in this context. Conversely, urinary AFB1 levels were not found to be associated with pediatric CD risk. These findings highlight ZEN as a specific environmental exposure that may contribute to the pathogenesis of Crohn’s disease in children, possibly through its estrogenic and immunomodulatory properties. While these results are significant, further large-scale, prospective, and mechanistic studies are essential to confirm these associations, elucidate the underlying biological pathways, and explore the potential for targeted interventions to mitigate ZEN exposure and its impact on pediatric CD. Declarations Ethical approval This study protocol was approved by the ethics committee of the forth affiliated hospital of Soochow university (Approval number: 180005). All research activities, including sample collection and assessments, were conducted in accordance with the ethical principles of the Declaration of Helsinki (2013 revision). Participants were fully informed about the study’s objectives and procedures, and all provided voluntary written informed consent prior to enrollment. Competing interests The authors declare no competing interests. Funding This work was supported by the Suzhou Gusu Health Talent Program (GSWS2020059) Author Contribution Jie Wang and Bin Xue contributed equally to the design of the study and the direction of its implementation, including supervision of the field activities, quality assurance, and control. Jian Huang supervised the field activities. Kai Zheng were responsible for sample collection. Zhongqin Jin helped conduct the literature review and prepare the Materials and methods and the Discussion sections of the text. Ning Shen and Jie Zhang designed the study’s analytic strategy and conducted the data analysis. All authors read and approved the final manuscript. Data Availability Data is provided within the manuscript. References Torres, J., Mehandru, S. & Colombel, J. F. Peyrin-Biroulet, L. Crohn's disease. Lancet 389 , 1741–1755 (2017). Jairath, V. & Feagan, B. G. Global burden of inflammatory bowel disease. Lancet Gastroenterol. Hepatol. 5 , 2–3 (2020). Khan, R., Kuenzig, M. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6711624","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":485028074,"identity":"2d43cd72-39ba-4f7f-9b6a-b8d1e639d0c7","order_by":0,"name":"Kai Zheng","email":"","orcid":"","institution":"The Forth Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zheng","suffix":""},{"id":485028076,"identity":"965f6379-a0d1-4981-9487-dd8a0e22757b","order_by":1,"name":"Ning Shen","email":"","orcid":"","institution":"Kangda College of Nanjing Medical University Affiliated Nantong Mental Health Center","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Shen","suffix":""},{"id":485028079,"identity":"ae461949-1dc9-4989-8f1a-c756c51323da","order_by":2,"name":"Zhongqin Jin","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Zhongqin","middleName":"","lastName":"Jin","suffix":""},{"id":485028081,"identity":"f7a94254-f782-4e2c-aaee-099ac7b26e75","order_by":3,"name":"Jie Zhang","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhang","suffix":""},{"id":485028082,"identity":"aa1b656d-1e07-41bf-aa9a-aee37ff67e0c","order_by":4,"name":"Jian Huang","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Huang","suffix":""},{"id":485028083,"identity":"7d2c2b1f-b875-46fe-9090-3154f5e01102","order_by":5,"name":"Bin Xue","email":"","orcid":"","institution":"Children’s Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Xue","suffix":""},{"id":485028086,"identity":"1bc0a41f-c475-463d-a505-0a98ef20c849","order_by":6,"name":"Jie Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYFCCAyCCjYefmfnwA5K0yEm2s6UZkGSXscF5HgUJopSaM55Ok2Bs40vcfJiHwYChxiaaoBbLhrPbgFrYErcd5j3wgOFYWm4DIS0GB0BatoG08CUYMDYcJkHL5mYeAwmStBgbMJOgZbMF4z82OYnDwEBOIMovN85uvMFw5hgPf//hww8+1NgQ1sIgcYCB+Q/DMQgngaByEOAHm1pDlNpRMApGwSgYoQAAeu0/vpZyCBoAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-21 02:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6711624/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6711624/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86769774,"identity":"e4aefe21-1233-472f-a48f-769b04e73ac0","added_by":"auto","created_at":"2025-07-15 11:35:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178923,"visible":true,"origin":"","legend":"\u003cp\u003eThe ROC analysis of the screen performance of Zearalenone and Aflatoxin B1\u003c/p\u003e\n\u003cp\u003eA, The ROC analysis of the screen performance of Zearalenone; B, The ROC analysis of the screen performance of Aflatoxin B1\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6711624/v1/9ba15da8be4dd5b943d93b0a.png"},{"id":100361066,"identity":"f2f15f96-f85c-48b7-8491-bea98ed3b57f","added_by":"auto","created_at":"2026-01-16 07:44:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":687368,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6711624/v1/793ebc41-b372-4385-bf9c-f6e0d09d0537.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The association between mycotoxin levels in urine of children and the risk of Crohn’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCrohn\u0026rsquo;s disease (CD) is a chronic, relapsing inflammatory condition characterized by transmural inflammation that can affect any part of the gastrointestinal tract, from the mouth to the anus\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The global incidence and prevalence of CD have been steadily increasing, particularly in newly industrialized countries and notably among children and adolescents\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Pediatric-onset CD often presents with a more severe disease course and significantly impacts growth, pubertal development, bone health, quality of life, and psychosocial well-being\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. While the exact etiology of CD remains incompletely understood, it is widely accepted to result from a complex interplay between genetic susceptibility, immune system dysregulation, and various environmental factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEnvironmental factors are gaining increasing attention in the pathogenesis of CD, as changes in environmental exposures are thought to underlie the rapid rise in incidence observed over recent decades, which cannot be solely explained by genetic shifts\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These factors encompass a wide range, including diet, smoking, antibiotic use, hygiene conditions, infections, and exposure to environmental contaminants\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Among dietary contaminants, mycotoxins which is the toxic secondary metabolites produced by fungi that commonly contaminate food staples represent a potential class of environmental triggers warranting investigation due to their ubiquitous nature and diverse biological effects\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study focuses on two prevalent mycotoxins: Aflatoxin B1 (AFB1) and Zearalenone (ZEN). AFB1, primarily produced by \u003cem\u003eAspergillus flavus\u003c/em\u003e and \u003cem\u003eAspergillus parasiticus\u003c/em\u003e, is a frequent contaminant of cereals (especially maize), oilseeds, spices, and nuts\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It is classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC) due to its potent hepatotoxicity and carcinogenicity, mediated through DNA adduct formation following metabolic activation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Beyond its carcinogenic potential, AFB1 has also been suggested to exert immunomodulatory effects, potentially altering immune cell function and cytokine production\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Zearalenone (ZEN), produced mainly by Fusarium species, is commonly found in maize, wheat, barley, and other grains\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. ZEN is structurally similar to endogenous estrogens and exhibits potent estrogenic activity by binding to estrogen receptors, classifying it as a mycoestrogen or xenoestrogen\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This endocrine-disrupting property can affect the reproductive system, but ZEN has also been shown to modulate immune responses, potentially influencing inflammatory processes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We chose to investigate AFB1 and ZEN because they are common dietary contaminants worldwide, and their respective toxicological profiles including potential immune modulation (AFB1, ZEN) and significant endocrine-disrupting activity (ZEN) suggest plausible mechanisms through which they might influence intestinal inflammation and CD risk.\u003c/p\u003e\u003cp\u003eDespite the potential relevance, research specifically investigating the association between exposure to AFB1 and ZEN and the risk of CD, particularly in pediatric populations, remains limited and inconclusive\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. While assessing exposure accurately is crucial, utilizing urinary biomarkers offers an objective measure of recent internal exposure, overcoming limitations associated with dietary recall methods\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, investigating the relationship between urinary levels of these specific mycotoxins and pediatric CD is necessary to clarify their potential role as environmental risk factors.\u003c/p\u003e\u003cp\u003eGiven this background, the primary objective of this case-control study was to investigate the association between urinary concentrations of AFB1 and ZEN and the risk of Crohn\u0026rsquo;s disease among Chinese children. We hypothesized that higher urinary levels of ZEN and/or AFB1 would be associated with an increased risk of developing CD in this population. A secondary objective was to evaluate the potential screening performance of urinary ZEN and AFB1 levels as biomarkers for discriminating between children with CD and healthy controls.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cp\u003eParticipants\u003c/p\u003e\u003cp\u003eThis study was conducted at the forth affiliated hospital of Soochow university between September 2021 and December 2022. The study protocol was approved by the ethics committee of the forth affiliated hospital of Soochow university (Approval Number: 180005). Written informed consent was obtained from the parents or legal guardians of all participants, and assent was obtained from children capable of understanding the study\u0026rsquo;s purpose.\u003c/p\u003e\u003cp\u003e\u003cem\u003eInclusion criteria for cases\u003c/em\u003e: Patients (aged 8 to 18 years) either newly diagnosed with CD (within 1 year) or those with established disease who had relapsed within 3 months according to Consensus Guidelines for the Diagnosis and Treatment of Inflammatory Bowel Disease\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e were eligible for inclusion as cases (n\u0026thinsp;=\u0026thinsp;32). \u003cem\u003eExclusion criteria for cases\u003c/em\u003e: Patients with indeterminate colitis, co-existing significant gastrointestinal infections at the time of diagnosis, known genetic syndromes associated with IBD-like symptoms, or severe concomitant systemic diseases were excluded. Patients who had already started specific CD treatments like biologics or extensive immunosuppression before sample collection was also excluded. \u003cem\u003eInclusion criteria for controls\u003c/em\u003e: Healthy children (n\u0026thinsp;=\u0026thinsp;30) visiting the hospital during the same period for routine health check-ups or minor, non-gastrointestinal, non-immunological conditions (e.g., minor trauma, scheduled vaccinations) were recruited as controls. Controls were selected to be generally comparable in age range to the cases. \u003cem\u003eExclusion criteria for controls\u003c/em\u003e: Children with any history of chronic gastrointestinal disease, inflammatory conditions, known immune deficiencies, malabsorption syndromes, chronic liver or kidney disease, or recent use of medications known to affect gut function or mycotoxin metabolism.\u003c/p\u003e\u003cp\u003eClinical Assessments\u003c/p\u003e\u003cp\u003eDemographic information, including age and gender, was collected for all participants through standardized questionnaires administered to the parents/guardians and/or review of medical records. For CD cases, relevant clinical data pertaining to diagnosis confirmation were extracted from medical records.\u003c/p\u003e\u003cp\u003eSample Collection and Preparation\u003c/p\u003e\u003cp\u003eSpot urine samples (first morning void) were collected from all participants using sterile, single-use containers. Immediately after collection, samples were centrifuged at 3000 rpm for 10 minutes at 4\u0026deg;C to remove cellular debris. The supernatant was carefully transferred into labeled polypropylene tubes, aliquoted, and stored frozen at -80\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003eAnalysis of Urinary Mycotoxins by HPLC-MS/MS\u003c/p\u003e\u003cp\u003eUrinary concentrations of AFB1 and ZEN were quantified using a validated HPLC-MS/MS method\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, urine samples (10mL) underwent an enzymatic hydrolysis step using 10ul β-glucuronidase/arylsulfatase (\u0026ge;\u0026thinsp;100,000 units/mL, Sinopharm, Shanghai, China) to release conjugated forms of the mycotoxins. The mixture was then incubated at 25\u0026deg;C overnight in a water bath incubator (YIHENG, Shanghai, China) to complete the enzymatic hydrolysis. After enzymatic hydrolysis, the sample was extracted using an SPE column (C18 ENVI, 0.25 g). The extract was eluted with methanol (2mL). Subsequently, the solvent was evaporated to dryness using a Termovap sample concentrator (Baojing, Zhengzhou, Henan, China) operated within a biological safety cabinet. Drying was achieved under a stream of high-purity nitrogen gas (\u0026ge;\u0026thinsp;99.9%, Baoshan Praxair, Shanghai, China) delivered at a flow rate of 3L/min (outlet pressure set to 0.3 MPa). Finally, methanol (100 \u0026micro;L) was used to redissolve the analyte. Fifty microliters of the analyte solution was transferred using a 100\u0026micro;L microsyringe (Shimadzu, Kyoto, Japan) to a 1.5mL autosampler vial (Shimadzu, Kyoto, Japan) for subsequent injection and analysis of mycotoxin levels. Detection and quantification were performed using a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode, monitoring specific precursor-to-product ion transitions for AFB1 and ZEN. The 1 \u0026micro;g/mL standard working solutions of AFB1 and ZEN (\u0026ge;\u0026thinsp;99%, TargetMol, Wellesley Hills, MA, USA) were prepared with methanol (\u0026ge;\u0026thinsp;99.9%, Supelco, Bellefonte, PA, USA) as solvent. Quality control measures, including procedural blanks, spiked samples, and replicate analyses, were incorporated throughout the analytical process to ensure data reliability.\u003c/p\u003e\u003cp\u003eStatistical Analyses\u003c/p\u003e\u003cp\u003eAll statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize participant characteristics. Differences in demographic characteristics and urinary mycotoxin levels between the case and control groups were assessed using the Mann-Whitney U test for continuous variables and the Pearson Chi-square (χ\u0026sup2;) test.\u003c/p\u003e\u003cp\u003eTo evaluate the association between urinary mycotoxin concentrations and the risk of CD, binary logistic regression analysis was performed. Log-transformed concentrations of urinary AFB1 and ZEN were then categorized into quartiles based on the distribution in the total population. The lowest two quartiles (Q1\u0026thinsp;+\u0026thinsp;Q2) were combined and used as the reference category. ORs and their corresponding 95% CIs were calculated for the third quartile (Q3) and fourth quartile (Q4) relative to the reference group (Q1\u0026thinsp;+\u0026thinsp;Q2). The logistic regression models were adjusted for age as a potential confounder. ROC curve analysis was conducted to assess the discriminatory ability of urinary ZEN and AFB1 concentrations to distinguish between CD cases and healthy controls. The AUC with its 95% CI was calculated for each mycotoxin. A two-sided P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant for all analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDemographic characteristics of study participants\u003c/p\u003e\u003cp\u003eA total of 62 children were enrolled in this study, comprising 32 children diagnosed with CD cases and 30 healthy children (controls). The demographic characteristics of the participants are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age of the CD cases (12 years, IQR: 11\u0026ndash;14 years) was significantly younger than that of the control group (13 years, IQR: 13\u0026ndash;14 years) (Z = -2.17, P\u0026thinsp;=\u0026thinsp;0.030). There was no statistically significant difference in gender distribution between the case group (17 males, 53.10%) and the control group (15 males, 50.00%) (χ\u0026sup2; = 0.06, P\u0026thinsp;=\u0026thinsp;0.806).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe demographics for cases and control\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eCase (n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZ/χ2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25th\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedian\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75th\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25th\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMedian\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e75th\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e-2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.030\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e15(50.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003e17(53.10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.806\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAflatoxin B1 (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e-0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.989\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZearalenone(pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e-5.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eComparison of urinary mycotoxin levels\u003c/p\u003e\u003cp\u003eUrinary concentrations of AFB1 and ZEN were compared between CD cases and controls, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. No significant difference was observed in the median urinary AFB1 concentrations between the CD cases (19.60 pg/ml, IQR: 14.07\u0026ndash;26.88 pg/ml) and the control group (19.78 pg/ml, IQR: 14.00-27.93 pg/ml) (Z = -0.01, P\u0026thinsp;=\u0026thinsp;0.989).\u003c/p\u003e\u003cp\u003eIn contrast, urinary ZEN concentrations were significantly higher in children with CD compared to healthy controls. The median urinary ZEN level in the case group was 0.33 pg/ml (IQR: 0.26\u0026ndash;0.36 pg/ml), which was substantially and statistically significantly higher than the median level of 0.16 pg/ml (IQR: 0.15\u0026ndash;0.21 pg/ml) found in the control group (Z = -5.73, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eAssociation between urinary mycotoxin concentrations and Crohn\u0026rsquo;s disease risk\u003c/p\u003e\u003cp\u003eThe associations between log-transformed urinary mycotoxin concentrations (categorized into quartiles, with Q1\u0026thinsp;+\u0026thinsp;Q2 combined as the reference) and the risk of CD were evaluated using logistic regression analysis, with results presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociations of mycotoxin concentrations with Crohn's disease (n\u0026thinsp;=\u0026thinsp;62).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMycotoxins\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCase(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOR*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95% C.I.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e Value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eAflatoxin B1 (Log transformed)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ1\u0026thinsp;+\u0026thinsp;Q2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15(50.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16(50.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7(23.30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9(28.10)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.31\u0026ndash;3.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8670\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8(26.70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7(21.90)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28\u0026ndash;3.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9940\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eZearalenone (Log transformed)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ1\u0026thinsp;+\u0026thinsp;Q2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25(83.30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5(15.60)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3(10.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15(46.90)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.54-351.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQ4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2(6.70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12(37.50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e62.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.31-541.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0002*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*These ORs were adjusted for age and gender.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor AFB1, after categorizing log-transformed concentrations, no significant association with CD risk was found. Compared to the reference group (Q1\u0026thinsp;+\u0026thinsp;Q2, 50.00% cases vs. 50.00% controls), children in the third quartile (Q3) of AFB1 exposure had an Odds Ratio (OR) of 1.12 (95% Confidence Interval [CI]: 0.31\u0026ndash;3.98, P\u0026thinsp;=\u0026thinsp;0.8670). Similarly, children in the fourth quartile (Q4) of AFB1 exposure showed an OR of 1.01 (95% CI: 0.28\u0026ndash;3.64, P\u0026thinsp;=\u0026thinsp;0.9940) for CD risk.\u003c/p\u003e\u003cp\u003eA strong, dose-dependent positive association was observed between higher urinary ZEN concentrations and the risk of CD. In the reference group (Q1\u0026thinsp;+\u0026thinsp;Q2), 83.30% were controls and 15.60% were cases. For children in the third quartile (Q3) of log-transformed ZEN exposure, the risk of CD was markedly increased, with an OR of 47.94 (95% CI: 6.54-351.77, P\u0026thinsp;=\u0026thinsp;0.0001) compared to the reference group. This association was even stronger for children in the highest quartile (Q4) of ZEN exposure, who had an OR of 62.93 (95% CI: 7.31-541.87, P\u0026thinsp;=\u0026thinsp;0.0002) for developing CD. These ORs were all adjusted for age and gender.\u003c/p\u003e\u003cp\u003eScreening performance of urinary mycotoxins for Crohn\u0026rsquo;s disease\u003c/p\u003e\u003cp\u003eROC curve analysis was performed to evaluate the ability of urinary ZEN and AFB1 concentrations to discriminate between children with CD and healthy controls. The results of this analysis are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The AUC for ZEN was 0.92 (95% CI: 0.85 to 1.00), indicating good discriminatory power. In contrast, the AUC for AFB1 was substantially lower at 0.50 (95% CI: 0.35 to 0.65), suggesting poor discriminatory ability, not significantly different from chance. The ROC curve for ZEN was positioned notably higher and to the left compared to the curve for AFB1, visually confirming its superior performance in distinguishing between the two groups. Based on the ZEN ROC curve, an optimal cut-off value of 0.205 pg/mL yielded a sensitivity of 100% and a specificity of 76.7%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the association between urinary levels of two common dietary mycotoxins, ZEN and AFB1, and the risk of Crohn\u0026rsquo;s disease in a pediatric cohort. The principal finding was a strong, significant, and dose-dependent positive association between elevated urinary ZEN concentrations and an increased risk of CD. In contrast, no significant association was observed between urinary AFB1 levels and CD risk. These results suggest that exposure to ZEN, but not necessarily all mycotoxins, may be an important environmental risk factor for pediatric CD, potentially linked to its estrogenic properties.\u003c/p\u003e\u003cp\u003eThe robust association between ZEN and pediatric CD warrants a detailed exploration of potential underlying biological mechanisms. ZEN is well-characterized as a potent mycoestrogen, structurally similar to endogenous estrogens, enabling it to bind to and activate estrogen receptors (ERs)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This estrogenic activity is a plausible link to CD pathogenesis through several pathways. Firstly, ZEN can exert significant immunomodulatory effects\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Estrogens are known to influence the balance of T helper (Th) cell subsets, such as Th1, Th2, and Th17, and modulate the production of various cytokines involved in inflammatory processes\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. ZEN, by mimicking estrogen, could dysregulate these critical immune responses within the gut. For instance, studies have shown ZEN can alter cytokine profiles and immune cell functions\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Secondly, ZEN exposure has been linked to the disruption of intestinal barrier integrity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Increased intestinal permeability (\u0026ldquo;leaky gut\u0026rdquo;) is a key feature in CD pathogenesis, allowing increased translocation of luminal antigens and triggering inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. ZEN has been demonstrated to compromise intestinal epithelial tight junctions and increase permeability in in vitro and in vivo models\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Thirdly, the gut itself is an estrogen-responsive organ, expressing ERα, ERβ, and G protein-coupled estrogen receptor 1 (GPER1)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. ZEN, acting through these receptors on intestinal epithelial cells, immune cells, or even enteric neurons, could directly modulate local inflammatory pathways. Finally, emerging evidence suggests that estrogens and xenoestrogens can influence the composition and function of the gut microbiome, which plays a crucial role in IBD\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Alterations in the gut microbiota by ZEN could further contribute to intestinal dysbiosis and inflammation. These interconnected mechanisms provide a biologically plausible framework for ZEN\u0026rsquo;s involvement in CD.\u003c/p\u003e\u003cp\u003eWhile AFB1 is a potent hepatotoxin and carcinogen\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and has been shown to possess some immunomodulatory properties\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, its primary toxicological impact might be less directed towards the specific immune dysregulation seen in CD compared to the estrogenic effects of ZEN. The main target organ for AFB1 toxicity is the liver, and its effects on gut immunity might be secondary or less pronounced.\u003c/p\u003e\u003cp\u003eComparing our findings with existing literature, studies on mycotoxins and IBD, particularly pediatric CD, are limited. It was reported that food-associated mycotoxins, including ZEN, could be risk factors in individuals predisposed to chronic intestinal inflammatory diseases by affecting intestinal permeability and immune responses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Studies investigating endocrine-disrupting chemicals (EDCs) have also reported associations with IBD or immune dysregulation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, lending indirect support to our findings regarding ZEN. Our finding of a strong association for ZEN aligns with experimental evidence showing its pro-inflammatory and barrier-disrupting effects in intestinal models. Our study adds specific evidence regarding ZEN and AFB1 in a pediatric CD context, highlighting ZEN as a particularly relevant exposure. In-depth mechanistic studies, utilizing animal models of IBD, intestinal organoids, and in vitro cell culture systems, are crucial to delineate the precise molecular pathways through which ZEN impacts intestinal inflammation, barrier function, immune cell responses, and the gut microbiome.\u003c/p\u003e\u003cp\u003eSeveral limitations of this study should be considered. The case-control design inherently limits our ability to establish causality, and though biomarkers reduce recall bias, selection bias might still be present. The relatively small sample size (n\u0026thinsp;=\u0026thinsp;62) resulted in wide confidence intervals for the odds ratios and reduced the statistical power for more granular analyses, impacting the precision of our estimates. While a single spot urine sample provides an objective measure of recent exposure, it may not fully capture long-term or intermittent exposure patterns crucial for chronic disease development. Other potential confounders, such as detailed dietary intake patterns, specific genetic susceptibilities, socioeconomic status, and co-exposure to other environmental agents, were not assessed and could influence the observed associations. Finally, the study was conducted in a specific geographical population, which may limit the generalizability of the findings to other populations with different dietary habits, genetic backgrounds, or mycotoxin exposure profiles. Consequently, larger-scale prospective studies are warranted to confirm these findings and further elucidate the role of mycotoxin exposure in pediatric CD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides compelling evidence for a strong, dose-dependent positive association between elevated urinary ZEN concentrations and an increased risk of Crohn\u0026rsquo;s disease in the pediatric population investigated. The observed discriminatory ability of urinary ZEN further supports its potential relevance as a biomarker in this context. Conversely, urinary AFB1 levels were not found to be associated with pediatric CD risk. These findings highlight ZEN as a specific environmental exposure that may contribute to the pathogenesis of Crohn\u0026rsquo;s disease in children, possibly through its estrogenic and immunomodulatory properties. While these results are significant, further large-scale, prospective, and mechanistic studies are essential to confirm these associations, elucidate the underlying biological pathways, and explore the potential for targeted interventions to mitigate ZEN exposure and its impact on pediatric CD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003e This study protocol was approved by the ethics committee of the forth affiliated hospital of Soochow university (Approval number: 180005). All research activities, including sample collection and assessments, were conducted in accordance with the ethical principles of the Declaration of Helsinki (2013 revision). Participants were fully informed about the study\u0026rsquo;s objectives and procedures, and all provided voluntary written informed consent prior to enrollment.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Suzhou Gusu Health Talent Program (GSWS2020059)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJie Wang and Bin Xue contributed equally to the design of the study and the direction of its implementation, including supervision of the field activities, quality assurance, and control. Jian Huang supervised the field activities. Kai Zheng were responsible for sample collection. Zhongqin Jin helped conduct the literature review and prepare the Materials and methods and the Discussion sections of the text. Ning Shen and Jie Zhang designed the study\u0026rsquo;s analytic strategy and conducted the data analysis. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTorres, J., Mehandru, S. \u0026amp; Colombel, J. F. Peyrin-Biroulet, L. Crohn's disease. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e389\u003c/b\u003e, 1741\u0026ndash;1755 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJairath, V. \u0026amp; Feagan, B. G. Global burden of inflammatory bowel disease. \u003cem\u003eLancet Gastroenterol. 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Health\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, 1140786 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Crohn’s disease, Mycotoxin, Zearalenone, Aflatoxin B1","lastPublishedDoi":"10.21203/rs.3.rs-6711624/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6711624/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCrohn\u0026rsquo;s disease (CD), a chronic inflammatory bowel disease with increasing pediatric incidence, is influenced by environmental factors, including potential dietary contaminants like mycotoxins. This study investigated the association between urinary levels of two mycotoxins, zearalenone (ZEN) and aflatoxin B1 (AFB1), and CD risk in children. In a case-control study involving 32 children with CD and 30 healthy controls, urinary ZEN and AFB1 concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry. Urinary ZEN concentrations were significantly higher in CD cases compared to controls (median 0.33 pg/ml vs 0.16 pg/ml, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas no significant difference was observed for AFB1 levels (P\u0026thinsp;=\u0026thinsp;0.989). Age-adjusted logistic regression analysis revealed a strong, dose-dependent positive association between higher urinary ZEN quartiles and CD risk; compared to the lowest 50%, adjusted Odds Ratios for the third and fourth quartiles of ZEN were 47.94 (P\u0026thinsp;=\u0026thinsp;0.0001) and 62.93 (P\u0026thinsp;=\u0026thinsp;0.0002), respectively. No significant association was found between urinary AFB1 levels and CD risk. Receiver Operating Characteristic analysis showed ZEN had superior discriminatory ability for CD (Area Under Curve [AUC]\u0026thinsp;=\u0026thinsp;0.92) compared to AFB1 (AUC\u0026thinsp;=\u0026thinsp;0.50). These findings demonstrate a strong positive association between elevated urinary ZEN levels and pediatric CD risk, suggesting ZEN exposure, potentially linked to its estrogenic properties, as an environmental risk factor. Conversely, urinary AFB1 was not associated with CD risk in this cohort, highlighting the potential role of specific mycotoxins in pediatric CD pathogenesis and warranting further investigation.\u003c/p\u003e","manuscriptTitle":"The association between mycotoxin levels in urine of children and the risk of Crohn’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 11:35:49","doi":"10.21203/rs.3.rs-6711624/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b19b05e-4761-4294-b188-ffcff25a7fad","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51485398,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases"},{"id":51485399,"name":"Earth and environmental sciences/Natural hazards"}],"tags":[],"updatedAt":"2026-01-12T04:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 11:35:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6711624","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6711624","identity":"rs-6711624","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}