Cell-Type-Dependent Differential Cytotoxic and Genotoxic Effects: Comprehensive Mechanistic Insights of L-Ascorbic Acid on Healthy Lymphocytes and HL-60 Cancer Cells

l-Ascorbic acid exhibits paradoxical behavior as both antioxidant and pro-oxidant in cancer treatment, with mechanisms and optimal dosing remaining unclear. This in vitro study investigated l-ascorbic acid's effects on healthy lymphocytes and HL-60 leukemia cells using concentrations of 0.5–2 mg/mL for 6 and 24 h. Analyses included cell viability, gene expression, DNA damage, mutagenicity, and oxidative stress markers. The results demonstrated a dose-dependent increase in the metabolic activity of HL-60 cells, with a ~ sixfold increase observed at 2 mg/mL (p ≤ 0.001), and opposing modulation of FAS/catalase gene expression between the two cell types. In healthy lymphocytes, both genes were suppressed with increasing exposure, while HL-60 cells exhibited significant early induction (29-fold for FAS and 47-fold for catalase, p ≤ 0.001). l-Ascorbic acid concentrations (0.5–2 mg/mL) exhibited no significant genotoxicity in healthy cells, increased antioxidant status in healthy lymphocytes, and elevated oxidative stress (a 1.4-fold increase in the oxidative stress index at 24 h, p ≤ 0.001) in HL-60 cells. Ames testing confirmed non-mutagenicity, while plasmid analysis revealed bimodal effects, being pro-oxidant at an intermediate dose and protective at a high dose. These findings suggest that l-ascorbic acid requires cell-type-specific application in hematological malignancies and may offer a therapeutic window in leukemia treatment protocols, providing a foundation for optimizing combination strategies with conventional chemotherapeutics.