MODULATION OF IRON HOMEOSTASIS BY BROCCOLI (BRASSICA OLERACEA L.) SPROUTS EXTRACT IN AML12 CELLS

Sulforaphane (SFN) is a bioactive compound that modulates nuclear factor erythroid 2 (Nrf2), which activates genes related to iron homeostasis. Broccoli is known for its antioxidant and anti-inflammatory effects, mainly attributed to SFN. SFN concentrations are high in broccoli sprouts. This study investigated the effects of broccoli sprout extracts on cellular iron metabolism in AML12 cells. After germination, SFN concentrations in aqueous sprout extracts were determined by HPLC [3-day sprouts: 353.2μg/ml of SFN; 7-day sprouts: 225.7μg/ml of SFN] and used to standardize the extracts to provide final SFN concentrations of 2 and 50 μM. AML12 cells were treated with isolated SFN (2 and 50 μM) or aqueous extracts of three-day-old (BSE-3D) and seven-day-old (BSE-7D) sprouts, standardized to provide 2 or 50 μM SFN. The expression of hepcidin, ferroportin (FPN), ferritin (FTH1), divalent metal transporter 1 (DMT1), and transferrin receptor (TFR2) was evaluated by qPCR, Western blotting, ELISA, or immunocytochemistry. Experiments were performed in three independent replicates and analyzed using Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test (p < 0.05). Treatments with 50 μM isolated SFN and BSE-3D decreased FPN expression by 87% and 70%, respectively, compared with the control group, whereas BSE-3D (50 μM) increased ferritin expression by 185% compared with the control group, with values of 0.698 ± 0.11 and 0.245 ± 0.03, respectively. Increased ferritin expression, consistent with enhanced cellular iron storage, was accompanied by reduced expression of DMT1, TFR2, and hepcidin genes. Immunocytochemistry supported these findings, showing reduced FPN expression. ELISA showed no significant differences in hepcidin secretion. ROS production increased by 79% and 35% compared with the control group following treatment with isolated SFN and BSE-3D (50 μM), respectively. These treatments also reduced cell viability to 45.6 ± 15.0% and 62.98 ± 11.3% of the control, respectively. These findings suggest that SFN and BSE-3D modulate iron-homeostasis-related pathways, characterized by increased ferritin, reduced FPN, and increased ROS. Together, these changes are consistent with increased cellular iron storage and suggest a relationship between SFN-induced modulation of iron homeostasis, oxidative status, and cell viability.