{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T00:01:44Z","timestamp":1785283304046,"version":"3.55.0"},"reference-count":33,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T00:00:00Z","timestamp":1785196800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T00:00:00Z","timestamp":1785196800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Applied Toxicology"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Areca nut consumption is a primary risk factor for oral squamous cell carcinoma (OSCC), inducing a distinct cell death phenotype characterized by nuclear pyknosis and concurrent membrane compromise. While the morphological hallmarks of this process are documented, its underlying metabolic trajectory remains poorly defined. This study integrates morphological characterization with longitudinal\n                    <jats:sup>1<\/jats:sup>\n                    H NMR metabolomics to investigate the biochemical state transitions in OC2 oral cancer cells treated with areca nut extract (ANE). Morphological analysis confirmed rapid nuclear condensation, with diameters reducing from 20.0\u2009to 12.1\u2009\u03bcm, and a hybrid membrane phenotype defined by simultaneous phosphatidylserine externalization and propidium iodide uptake. These changes were accompanied by a significant escalation in autophagic flux, evidenced by an LC3\u2010II\/I ratio of 4.08. Longitudinal metabolomic profiling identified an acute metabolic redirection within 1\u2009h of exposure, dominated by significant surges in acetate (5.26\u2010fold) and lactate (3.72\u2010fold). This early stress phase transitioned into a terminal state by 12\u2009h, characterized by the systemic depletion of nitrogen metabolism (glutamine, glutamate, and leucine) and membrane precursors (\n                    <jats:italic>O<\/jats:italic>\n                    \u2010phosphocholine). Multivariate modeling via RM\u2010ASCA+ isolated a significant treatment\u2010specific effect accounting for 29.88% of the total variance (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009=\u20090.001), statistically distinguishing ANE\u2010induced death from classical apoptosis, necrosis, and autophagy. These findings characterize the ANE\u2010induced phenotype as a discrete metabolic state transition, pyknotic necrosis, driven by a maladaptive autophagic process and precipitous resource exhaustion. This study provides a rigorous biochemical framework for understanding the unique cellular pathology associated with areca nut\u2013induced cytotoxicity.\n                  <\/jats:p>","DOI":"10.1002\/jat.70370","type":"journal-article","created":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T23:55:33Z","timestamp":1785282933000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated Morphological and Longitudinal\n                    <sup>1<\/sup>\n                    H NMR Metabolomic Characterization of Areca Nut\u2013Induced Pyknotic Necrosis"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7320-7876","authenticated-orcid":false,"given":"Chung\u2010Ying","family":"Yu","sequence":"first","affiliation":[{"name":"Center for Nano Bio\u2010Detection National Chung Cheng University  Chiayi Taiwan"},{"name":"Department of Biomedical Sciences National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-6787-6975","authenticated-orcid":false,"given":"Tzu\u2010Teng","family":"Tseng","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheng\u2010Fen","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-3474-5548","authenticated-orcid":false,"given":"Yu\u2010Hsien","family":"Tseng","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng\u2010Yen","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6086-4169","authenticated-orcid":false,"given":"Ssu\u2010Ting","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1621-8082","authenticated-orcid":false,"given":"Hau\u2010Ren","family":"Chen","sequence":"additional","affiliation":[{"name":"Center for Nano Bio\u2010Detection National Chung Cheng University  Chiayi Taiwan"},{"name":"Department of Biomedical Sciences National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9780-4982","authenticated-orcid":false,"given":"Eugene C.","family":"Lin","sequence":"additional","affiliation":[{"name":"Center for Nano Bio\u2010Detection National Chung Cheng University  Chiayi Taiwan"},{"name":"Department of Chemistry and Biochemistry National Chung Cheng University  Chiayi Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,28]]},"reference":[{"issue":"11","key":"e_1_2_12_2_1","doi-asserted-by":"crossref","first-page":"2692","DOI":"10.1038\/nprot.2007.376","article-title":"Metabolic Profiling, Metabolomic and Metabonomic Procedures for NMR Spectroscopy of Urine, Plasma, Serum and Tissue Extracts","volume":"2","author":"Beckonert O.","year":"2007","journal-title":"Nature Protocols"},{"issue":"9","key":"e_1_2_12_3_1","doi-asserted-by":"crossref","first-page":"1527","DOI":"10.1093\/carcin\/22.9.1527","article-title":"Areca Nut Extract and Arecoline Induced the Cell Cycle Arrest but Not Apoptosis of Cultured Oral KB Epithelial Cells: Association of Glutathione, Reactive Oxygen Species and Mitochondrial Membrane Potential","volume":"22","author":"Chang M. C.","year":"2001","journal-title":"Carcinogenesis"},{"issue":"2","key":"e_1_2_12_4_1","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/S1368-8375(98)00101-8","article-title":"Primary Oral Squamous Cell Carcinoma: An Analysis of 703 Cases in Southern Taiwan","volume":"35","author":"Chen Y. K.","year":"1999","journal-title":"Oral Oncology"},{"issue":"4","key":"e_1_2_12_5_1","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1016\/j.jds.2020.05.007","article-title":"Mechanistic and Compositional Studies of the Autophagy\u2010Inducing Areca Nut Ingredient","volume":"15","author":"Chiu C. T.","year":"2020","journal-title":"Journal of Dental Sciences"},{"issue":"1","key":"e_1_2_12_6_1","doi-asserted-by":"crossref","first-page":"221","DOI":"10.21873\/anticanres.13943","article-title":"Chronic Stimulation of the Autophagy\u2010Inducing Ingredient of Areca Nut Promotes Tumor Growth In\u00a0Vivo Through up\u2010Regulation of Tumoral Autophagy","volume":"40","author":"Chiu C. 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Application in 1H NMR Metabonomics","volume":"78","author":"Dieterle F.","year":"2006","journal-title":"Analytical Chemistry"},{"key":"e_1_2_12_11_1","doi-asserted-by":"crossref","first-page":"31520","DOI":"10.1038\/srep31520","article-title":"Identification of Salivary Metabolomic Biomarkers for Oral Cancer Screening","volume":"6","author":"Ishikawa S.","year":"2016","journal-title":"Scientific Reports"},{"key":"e_1_2_12_12_1","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1016\/j.toxrep.2014.10.018","article-title":"Areca Nut Extracts Exert Different Effects in Oral Cancer Cells Depending on Serum Concentration: A Clue to the Various Oral Alterations in Betel Quid Chewers","volume":"1","author":"Ji W. T.","year":"2014","journal-title":"Toxicology Reports"},{"issue":"5","key":"e_1_2_12_13_1","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0063295","article-title":"Areca Nut Extract Induces Pyknotic Necrosis in Serum\u2010Starved Oral Cells via Increasing Reactive Oxygen Species and Inhibiting GSK3beta: An Implication for Cytopathic Effects in Betel Quid Chewers","volume":"8","author":"Ji W. T.","year":"2013","journal-title":"PLoS ONE"},{"issue":"1","key":"e_1_2_12_14_1","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1007\/s11306-011-0350-z","article-title":"State\u2010Of\u2010The Art Data Normalization Methods Improve NMR\u2010Based Metabolomic Analysis","volume":"8","author":"Kohl S. 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