Beyond phototoxicity: The dark side of new methylene blue on mitochondrial and cellular bioenergetics

New methylene blue (NMB) is a phenothiazine dye with recognized photodynamic properties. This study evaluated both light-dependent and intrinsic (light-independent) effects of NMB on mitochondrial function, hepatic metabolism, and cell viability using three models: isolated rat liver mitochondria, ex vivo perfused rat liver, and HepG2 cells. In isolated mitochondria, NMB uncoupled oxidative phosphorylation, decreasing the respiratory control (RC) and ADP/O ratios, even without irradiation. It increased inner membrane permeability, inferred from enhanced oxidation of exogenous NADH, and inhibited mitochondrial swelling. Red-light irradiation exacerbated these effects, leading to inhibition of respiratory chain complexes and F_oF₁-ATP synthase, along with reduced catalase and glutathione reductase activities and depletion of GSH. Oxidative damage was confirmed by elevated lipid peroxidation and protein carbonylation. In perfused liver, NMB disrupted oxygen consumption linked to ATP synthesis and impaired energy-dependent processes such as gluconeogenesis and ammonia detoxification, particularly under fasting. These effects correlated with decreased ATP/ADP and ATP/AMP ratios, potentially impairing pyruvate carboxylase and carbamoyl phosphate synthetase I. In livers from fed rats, NMB stimulated glycolysis and glycogenolysis, while in fasting conditions it also enhanced fructolysis—likely as a compensatory response to mitochondrial dysfunction. Additionally, NMB-mediated NADH oxidation may raise NAD⁺ availability and support citric acid cycle activity. Membrane destabilization and enzyme leakage occurred even without light, indicating strong intrinsic cytotoxicity. Red-light exposure further intensified hepatic damage, likely due to in situ activation of the photosensitizer. In HepG2 cells, NMB reduced cell viability in a concentration-dependent manner, with no significant difference between irradiated and non-irradiated conditions—highlighting a pronounced dark toxicity. These results demonstrate that NMB impairs mitochondrial function and hepatic metabolism through both photodynamic and non-photodynamic mechanisms. Given the consistent toxicity across all models, including in the absence of light, NMB's therapeutic potential as a photosensitizer appears limited, warranting careful toxicological assessment before clinical application.