Integrated Morphological and Longitudinal ¹ H NMR Metabolomic Characterization of Areca Nut–Induced Pyknotic Necrosis

ABSTRACT 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 1 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 to 12.1 μm, 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‐II/I ratio of 4.08. Longitudinal metabolomic profiling identified an acute metabolic redirection within 1 h of exposure, dominated by significant surges in acetate (5.26‐fold) and lactate (3.72‐fold). This early stress phase transitioned into a terminal state by 12 h, characterized by the systemic depletion of nitrogen metabolism (glutamine, glutamate, and leucine) and membrane precursors ( O ‐phosphocholine). Multivariate modeling via RM‐ASCA+ isolated a significant treatment‐specific effect accounting for 29.88% of the total variance ( p = 0.001), statistically distinguishing ANE‐induced death from classical apoptosis, necrosis, and autophagy. These findings characterize the ANE‐induced 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–induced cytotoxicity.