{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T16:19:56Z","timestamp":1787588396441,"version":"build-2736575974"},"reference-count":35,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T00:00:00Z","timestamp":1759104000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Food Safety Commission, Cabinet Office, Government of Japan","award":["JPCAFSC20212102"],"award-info":[{"award-number":["JPCAFSC20212102"]}]},{"DOI":"10.13039\/501100003478","name":"Ministry of Health, Labour and Welfare of Japan","doi-asserted-by":"crossref","award":["22KD1003"],"award-info":[{"award-number":["22KD1003"]}],"id":[{"id":"10.13039\/501100003478","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003478","name":"Ministry of Health, Labour and Welfare of Japan","doi-asserted-by":"crossref","award":["24KD1002"],"award-info":[{"award-number":["24KD1002"]}],"id":[{"id":"10.13039\/501100003478","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["23K09652"],"award-info":[{"award-number":["23K09652"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Nishimura International Scholarship Foundation, Japan"},{"name":"Association for Promotion of Research on Risk Assessment, Japan"},{"name":"Ichikawa International Scholarship Foundation"}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Toxics"],"abstract":"<jats:p>The urinary bladder is a primary target organ for environmental toxicants such as arsenic. The objects of this study were two-fold. First, we constructed a novel 3D urinary bladder mucosa model (3D-UBMM) composed of an overlying epithelium and a supporting subepithelial layer. Primary human bladder urothelial and fibroblast cells were immortalized by introducing the human CDK4R24C and TERT genes. The construction of the 3D-UBMM involved incorporating immortalized fibroblast cells into a collagen raft, while immortalized urothelial cells were cultured at the air-liquid interface. This 3D-UBMM closely resembles the human bladder epithelium in terms of morphology and marker protein expression, including uroplakin 1b, P63, and cytokeratin 5. Second, using the 3D-UBMM we investigated the cytotoxicity of sodium arsenite (iAsIII) and dimethylarsenic acid (DMAV). Exposure to iAsIII and DMAV resulted in increased urothelial necrosis, increased \u03b3-H2AX-positive cells, and reduced P63-positive cells, all in a dose\u2013response manner. These findings affirm that this novel 3D-UBMM resembles the human bladder epithelium and offers a practical in vitro model for evaluating bladder toxicants and carcinogens, identifying mechanisms of carcinogenesis, and supporting hazard identification and risk assessment.<\/jats:p>","DOI":"10.3390\/toxics13100828","type":"journal-article","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T09:12:23Z","timestamp":1759223543000},"page":"828","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Construction of a Novel 3D Urinary Bladder Mucosa Model and Its Application in Toxicity Assessment of Arsenicals"],"prefix":"10.3390","volume":"13","author":[{"given":"Runjie","family":"Guo","sequence":"first","affiliation":[{"name":"Department of Environmental Risk Assessment, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, 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Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guiyu","family":"Qiu","sequence":"additional","affiliation":[{"name":"Department of Environmental Risk Assessment, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9274-2869","authenticated-orcid":false,"given":"Kwanchanok","family":"Praseatsook","sequence":"additional","affiliation":[{"name":"Department of Environmental Risk Assessment, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yurina","family":"Kawamura","sequence":"additional","affiliation":[{"name":"Department of Environmental Risk Assessment, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1149-1450","authenticated-orcid":false,"given":"Anna","family":"Kakehashi","sequence":"additional","affiliation":[{"name":"Department of Molecular Pathology, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ikue","family":"Noura","sequence":"additional","affiliation":[{"name":"Department of Molecular Pathology, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoli","family":"Xie","sequence":"additional","affiliation":[{"name":"Department of Toxicology, School of Public Health, Southern Medical University (Guangdong Provincial Key Laboratory of Tropical Disease Research), Guangzhou 510515, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9362-4290","authenticated-orcid":false,"given":"Hideki","family":"Wanibuchi","sequence":"additional","affiliation":[{"name":"Department of Environmental Risk Assessment, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"},{"name":"Department of Molecular Pathology, Osaka Metropolitan University Graduate School of Medicine, Osaka 554-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1093\/bmb\/ldr003","article-title":"Tissue Engineering of Human Bladder","volume":"97","author":"Atala","year":"2011","journal-title":"Br. Med. Bull."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1038\/nature09851","article-title":"Hedgehog\/Wnt Feedback Supports Regenerative Proliferation of Epithelial Stem Cells in Bladder","volume":"472","author":"Shin","year":"2011","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1111\/bcpt.12626","article-title":"The Role of the Mucosa in Normal and Abnormal Bladder Function","volume":"119","author":"Fry","year":"2016","journal-title":"Basic Clin. Pharmacol. Toxicol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Du, Y.H., Miao, W.H., Jiang, X., Cao, J., Wang, B., Wang, Y., Yu, J., Wang, X.Z., and Liu, H.T. (2021). The Epithelial to Mesenchymal Transition Related Gene Calumenin Is an Adverse Prognostic Factor of Bladder Cancer Correlated With Tumor Microenvironment Remodeling, Gene Mutation, and Ferroptosis. Front. Oncol., 11.","DOI":"10.3389\/fonc.2021.683951"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1007\/978-1-0716-0648-3_5","article-title":"Organotypic 3D Skin Models: Human Epidermal Equivalent Cultures from Primary Keratinocytes and Immortalized Keratinocyte Cell Lines","volume":"2154","author":"Rikken","year":"2020","journal-title":"Methods Mol. Biol."},{"key":"ref_6","first-page":"229","article-title":"A Novel 3D Human Glioblastoma Cell Culture System for Modeling Drug and Radiation Responses","volume":"19","author":"Stevenson","year":"2017","journal-title":"Neuro-Oncology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5077","DOI":"10.1038\/s41467-020-18916-5","article-title":"Single-Cell RNA Sequencing Highlights the Role of Inflammatory Cancer-Associated Fibroblasts in Bladder Urothelial Carcinoma","volume":"11","author":"Chen","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_8","unstructured":"IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (2012). Arsenic, Metals, Fibres, and Dusts. IARC Monogr. Eval. Carcinog. Risks Hum., 100, 11."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.taap.2009.01.010","article-title":"Arsenic Exposure at Low-to-Moderate Levels and Skin Lesions, Arsenic Metabolism, Neurological Functions, and Biomarkers for Respiratory and Cardiovascular Diseases: Review of Recent Findings from the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh","volume":"239","author":"Chen","year":"2009","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.cotox.2019.05.002","article-title":"Inorganic Arsenic: A Nongenotoxic Threshold Carcinogen","volume":"14","author":"Cohen","year":"2019","journal-title":"Curr. Opin. Toxicol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"711","DOI":"10.3109\/10408444.2013.827152","article-title":"Evaluation of the Carcinogenicity of Inorganic Arsenic","volume":"43","author":"Cohen","year":"2013","journal-title":"Crit. Rev. Toxicol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1150","DOI":"10.1021\/tx020026z","article-title":"Possible Role of Dimethylarsinous Acid in Dimethylarsinic Acid-Induced Urothelial Toxicity and Regeneration in the Rat","volume":"15","author":"Cohen","year":"2002","journal-title":"Chem. Res. Toxicol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1093\/carcin\/23.8.1387","article-title":"Carcinogenicity of Dimethylarsinic Acid in Male F344 Rats and Genetic Alterations in Induced Urinary Bladder Tumors","volume":"23","author":"Wei","year":"2002","journal-title":"Carcinogenesis"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1080\/10408440500534230","article-title":"Methylated Arsenicals: The Implications of Metabolism and Carcinogenicity Studies in Rodents to Human Risk Assessment","volume":"36","author":"Cohen","year":"2006","journal-title":"Crit. Rev. Toxicol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.taap.2006.10.010","article-title":"Arsenic-Induced Bladder Cancer in an Animal Model","volume":"222","author":"Cohen","year":"2007","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.tox.2006.03.013","article-title":"Dimethylarsinic Acid: Results of Chronic Toxicity\/Oncogenicity Studies in F344 Rats and in B6C3F1 Mice","volume":"223","author":"Arnold","year":"2006","journal-title":"Toxicology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.tox.2013.01.015","article-title":"Effect of Dietary Treatment with Dimethylarsinous Acid (DMAIII) on the Urinary Bladder Epithelium of Arsenic (+3 Oxidation State) Methyltransferase (As3mt) Knockout and C57BL\/6 Wild Type Female Mice","volume":"305","author":"Dodmane","year":"2013","journal-title":"Toxicology"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Nishiwaki, M., Toyoda, M., Oishi, Y., Ishida, S., Horiuchi, S.-I., Makino-Itou, H., Kimura, T., Ohno, S.-i., Ohkura, T., and Enosawa, S. (2020). Immortalization of Human Hepatocytes from Biliary Atresia with CDK4R24C, Cyclin D1, and TERT for Cytochrome P450 Induction Testing. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-73992-3"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1093\/carcin\/bgu112","article-title":"A Human Cancer Xenograft Model Utilizing Normal Pancreatic Duct Epithelial Cells Conditionally Transformed with Defined Oncogenes","volume":"35","author":"Inagawa","year":"2014","journal-title":"Carcinogenesis"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2065","DOI":"10.1007\/s00204-024-03750-1","article-title":"Characterizing the Toxicological Responses to Inorganic Arsenicals and Their Metabolites in Immortalized Human Bladder Epithelial Cells","volume":"98","author":"Vachiraarunwong","year":"2024","journal-title":"Arch. Toxicol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1016\/j.ajpath.2011.10.036","article-title":"ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells","volume":"180","author":"Liu","year":"2012","journal-title":"Am. J. Pathol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1093\/toxsci\/kfz051","article-title":"Acetoaceto-o-Toluidide Enhances Cellular Proliferative Activity in the Urinary Bladder of Rats","volume":"169","author":"Okuno","year":"2019","journal-title":"Toxicol. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Nowacka, M., Ginter-Matuszewska, B., \u015awierczewska, M., Sterzy\u0144ska, K., Nowicki, M., and Januchowski, R. (2022). Effect of ALDH1A1 Gene Knockout on Drug Resistance in Paclitaxel and Topotecan Resistant Human Ovarian Cancer Cell Lines in 2D and 3D Model. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23063036"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"561","DOI":"10.31557\/APJCP.2022.23.2.561","article-title":"Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening","volume":"23","author":"Baru","year":"2022","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1152\/physrev.00041.2019","article-title":"The Urothelium: Life in a Liquid Environment","volume":"100","author":"Dalghi","year":"2020","journal-title":"Physiol. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"dev198184","DOI":"10.1242\/dev.198184","article-title":"Development, Regeneration and Tumorigenesis of the Urothelium","volume":"149","author":"Wiessner","year":"2022","journal-title":"Development"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1080\/15321819.2020.1845726","article-title":"Stratification of Urinary Bladder Carcinoma Based on Immunohistochemical Expression of CK5, CK14 and CK20","volume":"42","author":"Abdelwahed","year":"2021","journal-title":"J. Immunoass. Immunochem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"8105","DOI":"10.1073\/pnas.1221216110","article-title":"P63-Expressing Cells Are the Stem Cells of Developing Prostate, Bladder, and Colorectal Epithelia","volume":"110","author":"Pignon","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"111752","DOI":"10.1016\/j.ecoenv.2020.111752","article-title":"Different Mechanisms of Arsenic Related Signaling in Cellular Proliferation, Apoptosis and Neo-Plastic Transformation","volume":"208","author":"Medda","year":"2021","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1293\/tox.2020-0038","article-title":"Role of \u03b3-H2AX as a Biomarker for Detection of Bladder Carcinogens in F344 Rats","volume":"33","author":"Suzuki","year":"2020","journal-title":"J. Toxicol. Pathol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1093\/toxsci\/kfv192","article-title":"Early Detection of Genotoxic Urinary Bladder Carcinogens by Immunohistochemistry for \u03b3-H2AX","volume":"148","author":"Toyoda","year":"2015","journal-title":"Toxicol. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lovitt, C.J., Shelper, T.B., and Avery, V.M. (2018). Doxorubicin Resistance in Breast Cancer Cells Is Mediated by Extracellular Matrix Proteins. BMC Cancer, 18.","DOI":"10.1186\/s12885-017-3953-6"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1038\/s41385-022-00565-0","article-title":"The Urothelium: A Multi-Faceted Barrier against a Harsh Environment","volume":"15","author":"Jafari","year":"2022","journal-title":"Mucosal Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1093\/toxsci\/kfn184","article-title":"Effects of Inorganic Arsenic on the Rat and Mouse Urinary Bladder","volume":"106","author":"Suzuki","year":"2008","journal-title":"Toxicol. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1093\/toxsci\/kfq315","article-title":"Carcinogenic Effects of \u201cWhole-Life\u201d Exposure to Inorganic Arsenic in CD1 Mice","volume":"119","author":"Tokar","year":"2011","journal-title":"Toxicol. Sci."}],"container-title":["Toxics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2305-6304\/13\/10\/828\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T04:16:46Z","timestamp":1759292206000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2305-6304\/13\/10\/828"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,29]]},"references-count":35,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["toxics13100828"],"URL":"https:\/\/doi.org\/10.3390\/toxics13100828","relation":{},"ISSN":["2305-6304"],"issn-type":[{"value":"2305-6304","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,29]]}}}