Dynamic 99mTc-MIBI uptake and washout as functional biomarkers of glycyrrhizic acid-induced mitochondrial dysfunction and P-glycoprotein activity in HepG2 cells

OBJECTIVE: To investigate the effects of glycyrrhizic acid on HepG2 cell viability and the cellular uptake, retention, and washout kinetics of technetium-99m methoxyisobutylisonitrile (99mTc-MIBI), with particular emphasis on the potential involvement of mitochondrial function and P-glycoprotein (P-gp)-mediated efflux. METHODS: HepG2 cells were treated with glycyrrhizic acid at concentrations ranging from 0 to 100 μM, and cell viability was assessed using the MTT assay. Cellular 99mTc-MIBI uptake was evaluated over 5-120 min to characterize time-dependent tracer accumulation. Pulse-chase experiments were performed to assess intracellular tracer retention and washout over 30-180 min following tracer loading. The contribution of P-gp-mediated efflux was further investigated by assessing the effect of the P-gp inhibitor verapamil on intracellular 99mTc-MIBI accumulation in glycyrrhizic acid-treated cells. RESULTS: Glycyrrhizic acid reduced HepG2 cell viability in a concentration- and time-dependent manner, with an IC50 of approximately 60 μM after 48 h. In untreated cells, 99mTc-MIBI accumulation progressively increased and approached a plateau between 60 and 120 min. Glycyrrhizic acid treatment reduced intracellular tracer uptake in a concentration-dependent manner. Pulse-chase experiments demonstrated decreased intracellular tracer retention and increased washout following glycyrrhizic acid treatment. Pretreatment with verapamil partially increased intracellular 99mTc-MIBI accumulation compared with glycyrrhizic acid treatment alone, indicating that P-gp-mediated efflux may contribute to the reduced tracer retention observed after glycyrrhizic acid exposure. CONCLUSION: Glycyrrhizic acid alters 99mTc-MIBI uptake, intracellular retention, and washout kinetics in HepG2 cells. The observed changes are consistent with altered mitochondrial tracer sequestration and increased transporter-mediated efflux, although the present findings do not establish these mechanisms directly. Dynamic 99mTc-MIBI kinetics may therefore provide a functional approach for assessing cellular responses to glycyrrhizic acid, warranting further mechanistic and in-vivo validation.