{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:03:50Z","timestamp":1766707430629,"version":"3.48.0"},"reference-count":30,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,25]],"date-time":"2025-12-25T00:00:00Z","timestamp":1766620800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001807","name":"S\u00e3o Paulo Research Foundation","doi-asserted-by":"publisher","award":["2022\/03952-1"],"award-info":[{"award-number":["2022\/03952-1"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001807","name":"S\u00e3o Paulo Research Foundation","doi-asserted-by":"publisher","award":["2019\/21603-1"],"award-info":[{"award-number":["2019\/21603-1"]}],"id":[{"id":"10.13039\/501100001807","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Aflatoxins (AFs) are potent hepatotropic mycotoxins\u2014AFB1 being the best-characterized\u2014yet their ability to induce intrahepatic cholangiocarcinoma (iCCA) remains underexplored. Male Wistar rats received vehicle (controls; n = 5) or an AFB1-dominant AF mixture (AFB1 39.46 \u03bcg\/mL; AFB2 1.13 \u03bcg\/mL; AFG1 17.44 \u03bcg\/mL; AFG2 0.59 \u03bcg\/mL\u2014 n = 10) by daily gavage for 90 days, at a dose equivalent to 400 \u03bcg AFB1 per kg of diet. After 12 months, twelve iCCA tumors were resected and analyzed by histology (H&amp;E) and tissue-microarray-based immunohistochemistry (Cytokeratin-19, Hep Par-1, p53, Cyclin D1, Rb, \u03b2-catenin, and PCNA). Lesions predominantly showed glandular\/tubular architecture consistent with iCCA and were cytokeratin-19-positive and Hep Par-1-negative. Cell proliferation was high (PCNA \u2248 69%). p53 displayed nuclear accumulation in 83% of tumors. G1\/S control was perturbed, with cyclin D1 overexpression (67%), and Rb was positive in 58% of iCCA. Aberrant Wnt activation was rare (nuclear \u03b2-catenin in 8%). Subchronic exposure to an AFB1-dominant AF mixture in rats was associated with iCCA characterized by high proliferative activity, p53 accumulation, and disruption of G1\/S checkpoint components. These findings broaden the oncogenic spectrum of AFs and warrant genomic confirmation of AF mutational signatures.<\/jats:p>","DOI":"10.3390\/toxins18010014","type":"journal-article","created":{"date-parts":[[2025,12,25]],"date-time":"2025-12-25T23:52:27Z","timestamp":1766706747000},"page":"14","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Aflatoxin Mixture-Driven Intrahepatic Cholangiocarcinoma in Rats Involving G1\/S Checkpoint Dysregulation"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-4373-4783","authenticated-orcid":false,"given":"Vin\u00edcius Menezes","family":"Braga","sequence":"first","affiliation":[{"name":"Department of Pathology and Legal Medicine, Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, Av. Bandeirantes, 3900, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paulo Henrique Fernandes","family":"Pereira","sequence":"additional","affiliation":[{"name":"Department of Pathology and Legal Medicine, Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, Av. Bandeirantes, 3900, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6915-5615","authenticated-orcid":false,"given":"Let\u00edcia de Araujo","family":"Apolinario","sequence":"additional","affiliation":[{"name":"Department of Nursing in Hospital Care, Federal University of the Tri\u00e2ngulo Mineiro, R. Vig\u00e1rio Carlos, 100, Uberaba 38025-350, MG, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Deisy Mara Silva","family":"Longo","sequence":"additional","affiliation":[{"name":"Department of Pathology and Legal Medicine, Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, Av. Bandeirantes, 3900, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leandra Naira Zambelli","family":"Ramalho","sequence":"additional","affiliation":[{"name":"Department of Pathology and Legal Medicine, Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, Av. Bandeirantes, 3900, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4604-9702","authenticated-orcid":false,"given":"Sher","family":"Ali","sequence":"additional","affiliation":[{"name":"Department of Food Engineering, School of Animal Science and Food Engineering, University of S\u00e3o Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5779-5287","authenticated-orcid":false,"given":"Carlos Augusto Fernandes de","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Department of Food Engineering, School of Animal Science and Food Engineering, University of S\u00e3o Paulo, Av. Duque de Caxias Norte, 225, Pirassununga 13635-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7529-466X","authenticated-orcid":false,"given":"Fernando Silva","family":"Ramalho","sequence":"additional","affiliation":[{"name":"Department of Pathology and Legal Medicine, Ribeir\u00e3o Preto Medical School, University of S\u00e3o Paulo, Av. Bandeirantes, 3900, Ribeir\u00e3o Preto 14049-900, SP, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1038\/s41575-020-0310-z","article-title":"Cholangiocarcinoma 2020: The next horizon in mechanisms and management","volume":"17","author":"Banales","year":"2020","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1038\/s41572-021-00300-2","article-title":"Cholangiocarcinoma","volume":"7","author":"Brindley","year":"2021","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","unstructured":"International Agency for Research on Cancer (IARC) (2012). Aflatoxins. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, IARC."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S28","DOI":"10.1093\/toxsci\/kfq283","article-title":"Aflatoxin: A 50-year odyssey of mechanistic and translational toxicology","volume":"120","author":"Kensler","year":"2011","journal-title":"Toxicol. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5858","DOI":"10.1038\/sj.onc.1208732","article-title":"249(Ser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma","volume":"24","author":"Kirk","year":"2005","journal-title":"Oncogene"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1038\/350429a0","article-title":"Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa","volume":"350","author":"Bressac","year":"1991","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1475","DOI":"10.1101\/gr.220038.116","article-title":"Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors","volume":"27","author":"Huang","year":"2017","journal-title":"Genome Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1038\/s41586-020-1943-3","article-title":"The repertoire of mutational signatures in human cancer","volume":"578","author":"Alexandrov","year":"2020","journal-title":"Nature"},{"key":"ref_9","first-page":"271","article-title":"Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses","volume":"68","author":"Moore","year":"1982","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/S0015-6264(75)80011-3","article-title":"Induction of cholangiocarcinoma following treatment of a rhesus monkey with aflatoxin","volume":"13","author":"Tilak","year":"1975","journal-title":"Food Cosmet. Toxicol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1046\/j.1365-2559.1998.00522.x","article-title":"Hep Par 1 and selected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, combined tumours and metastatic carcinoma","volume":"33","author":"Leong","year":"1998","journal-title":"Histopathology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1097\/01.MP.0000052103.13730.20","article-title":"Hep par 1 antibody stain for the differential diagnosis of hepatocellular carcinoma: 676 tumors tested using tissue microarrays and conventional tissue sections","volume":"16","author":"Fan","year":"2003","journal-title":"Mod. Pathol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1016\/S0140-6736(21)00153-7","article-title":"Biliary tract cancer","volume":"397","author":"Valle","year":"2021","journal-title":"Lancet"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2800","DOI":"10.1200\/JCO.2007.15.5945","article-title":"Liver cancer stem cells","volume":"26","author":"Sell","year":"2008","journal-title":"J. Clin. Oncol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"a026179","DOI":"10.1101\/cshperspect.a026179","article-title":"Somatic TP53 mutations in the era of genome sequencing","volume":"6","author":"Hainaut","year":"2016","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1097\/00000478-200208000-00003","article-title":"Combined hepatocellular-cholangiocarcinoma: A histopathologic, immunohistochemical, and in situ hybridization study","volume":"26","author":"Tickoo","year":"2002","journal-title":"Am. J. Surg. Pathol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Choi, J.H., and Ro, J.Y. (2022). Combined Hepatocellular-Cholangiocarcinoma: An Update on Pathology and Diagnostic Approach. Biomedicines, 10.","DOI":"10.3390\/biomedicines10081826"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1212","DOI":"10.1016\/j.jhep.2021.01.035","article-title":"Combined hepatocellular-cholangiocarcinoma: An update","volume":"74","author":"Calderaro","year":"2021","journal-title":"J. Hepatol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1038\/s41467-018-03276-y","article-title":"Whole-Exome Sequencing Reveals the Origin and Evolution of Hepato-Cholangiocarcinoma","volume":"9","author":"Wang","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"932","DOI":"10.1016\/j.ccell.2019.04.007","article-title":"Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes","volume":"35","author":"Xue","year":"2019","journal-title":"Cancer Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"112263","DOI":"10.1016\/j.fct.2021.112263","article-title":"Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers","volume":"153","author":"Ramalho","year":"2021","journal-title":"Food Chem. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Khosla, D., Misra, S., Chu, P.L., Guan, P., Nada, R., Gupta, R., Kaewnarin, K., Ko, T.K., Heng, H.L., and Srinivasalu, V.K. (2024). Cholangiocarcinoma: Recent Advances in Molecular Pathobiology and Therapeutic Approaches. Cancers, 16.","DOI":"10.3390\/cancers16040801"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4199","DOI":"10.1158\/1078-0432.CCR-19-1292","article-title":"Retinoblastoma Loss in Cancer: Casting a Wider Net","volume":"25","author":"Choudhury","year":"2019","journal-title":"Clin. Cancer Res."},{"key":"ref_24","unstructured":"The Cancer Genome Atlas Research Network (2017). Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell, 169, 1327\u20131341.e23."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Lozano, E., Sanchon-Sanchez, P., Morente-Carrasco, A., Chinchilla-T\u00e1bora, L.M., Mauriz, J.L., Fern\u00e1ndez-Palanca, P., Marin, J.J.G., and Macias, R.I.R. (2023). Impact of Aberrant \u03b2-Catenin Pathway on Cholangiocarcinoma Heterogeneity. Cells, 12.","DOI":"10.3390\/cells12081141"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"900","DOI":"10.1038\/modpathol.3880409","article-title":"Altered Expression of \u03b2-Catenin without Genetic Mutation in Intrahepatic Cholangiocarcinoma","volume":"14","author":"Sugimachi","year":"2001","journal-title":"Mod. Pathol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"34","DOI":"10.20517\/2394-5079.2023.37","article-title":"Genomic Alterations in Intrahepatic Cholangiocarcinoma","volume":"9","author":"Young","year":"2023","journal-title":"Hepatoma Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2878","DOI":"10.1016\/j.celrep.2017.06.008","article-title":"Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles","volume":"19","author":"Farshidfar","year":"2017","journal-title":"Cell Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2839","DOI":"10.18632\/oncotarget.1943","article-title":"Multigene Mutational Profiling of Cholangiocarcinomas Identifies Actionable Molecular Subgroups","volume":"5","author":"Simbolo","year":"2014","journal-title":"Oncotarget"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Percie du Sert, N., Ahluwalia, A., Alam, S., Avey, M.T., Baker, M., Browne, W.J., Clark, A., Cuthill, I.C., Dirnagl, U., and Emerson, M. (2020). The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol., 18.","DOI":"10.1371\/journal.pbio.3000410"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/1\/14\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:00:17Z","timestamp":1766707217000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/18\/1\/14"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,25]]},"references-count":30,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["toxins18010014"],"URL":"https:\/\/doi.org\/10.3390\/toxins18010014","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,25]]}}}