Electrolyzed Water-Induced Activation of the Nrf2 Pathway and Antioxidant Responses

The nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor is a master regulator of cytoprotective antioxidant gene expression; nevertheless, the ability of electrolyzed water formulations and their metal salt combinations to modulate this pathway remains insufficiently characterized. This study aimed to investigate whether ASEA Redox Signaling Molecules Supplement (ARS) activates the Nrf2 pathway and whether selected metal salts enhance this effect. Nrf2 activation was quantified in AREc32 luciferase reporter cells; cytotoxicity was assessed via Alamar Blue assay. Extracellular glutathione (GSH) and related thiols were quantified by HPLC with fluorescence detection. Western blot analysis of HepG2 cells was performed to assess the expression of Nrf2-regulated cytoprotective proteins, including HO-1 and GCLC. ARS alone induced modest Nrf2 activation (1.78 ± 0.51-fold). Combination treatment with gold(III) chloride trihydrate (HAuCl₄.3H₂O) significantly enhanced activation (7.43 ± 1.15-fold), whereas the salt alone exhibited negligible activity. Zinc and copper salts exhibited moderate activation. Notably, ARS combined with gold(III) chloride trihydrate significantly elevated extracellular GSH (2.46 ± 2.73-fold) and upregulated HO-1 protein expression. Collectively, these data demonstrate that electrolyzed water formulations activate the Nrf2-ARE pathway in vitro, with marked potentiation by specific metal salts, particularly gold chloride trihydrate, implicating synergistic modulation of cellular antioxidant signaling.