The photosensitiser azure A disrupts mitochondrial bioenergetics through intrinsic and photodynamic effects

• Published in: Toxicology (Amsterdam) Vol. 455; p. 152766
• Main Authors: de Souza, Byanca Thais Lima, Klosowski, Eduardo Makiyama, Mito, Márcio Shigueaki, Constantin, Renato Polimeni, Mantovanelli, Gislaine Cristiane, Mewes, Juliana Morais, Bizerra, Paulo Francisco Veiga, da Silva, Fernanda Sayuri Itou, Menezes, Paulo Vinicius Moreira da Costa, Gilglioni, Eduardo Hideo, Utsunomiya, Karina Sayuri, Marchiosi, Rogério, dos Santos, Wanderley Dantas, Ferrarese-Filho, Osvaldo, Caetano, Wilker, de Souza Pereira, Paulo Cesar, Gonçalves, Renato Sonchini, Constantin, Jorgete, Ishii-Iwamoto, Emy Luiza, Constantin, Rodrigo Polimeni
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 15-05-2021
• Subjects: Adenosine Triphosphate - metabolism; Animals; ATP synthesis; Azure Stains - toxicity; Cell Survival - drug effects; Electron transfer; Energy Metabolism - drug effects; Hepatocytes - drug effects; Hepatocytes - pathology; Hepatotoxicity; Lipid Peroxidation - drug effects; Liver - drug effects; Liver - pathology; Male; Mitochondria, Liver - drug effects; Mitochondria, Liver - pathology; Oxygen Consumption - drug effects; Photooxidation; Photosensitisers; Protein Carbonylation - drug effects; Rats; Rats, Wistar; Reactive Oxygen Species - metabolism; Uncoupling; Uncoupling; Electron transfer; ATP synthesis; Photosensitisers; Photooxidation; Hepatotoxicity
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2021.152766

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•Mitochondria may serve as important cellular targets for azure A.•Azure A photoinactivates specific elements of the mitochondrial respiratory system.•Azure A impairs the mitochondrial bioenergetics even in the dark.•Azure A reduces cellular ATP levels and disrupts energy-linked hepatic metabolism.•Azure A has little potential to directly cause extensive cell viability reduction.
Azure A (AA) is a cationic molecule of the class of phenothiazines that has been applied in vitro as a photosensitising agent in photodynamic antimicrobial chemotherapy. It is a di-demethylated analogue of methylene blue (MB), which has been demonstrated to be intrinsically and photodynamically highly active on mitochondrial bioenergetics. However, as far as we know, there are no studies about the photodynamic effects of AA on mammalian mitochondria. Therefore, this investigation aimed to characterise the intrinsic and photodynamic acute effects of AA (0.540 μM) on isolated rat liver mitochondria, isolated hepatocytes, and isolated perfused rat liver. The effects of AA were assessed by evaluating several parameters of mitochondrial bioenergetics, oxidative stress, cell viability, and hepatic energy metabolism. The photodynamic effects of AA were assessed under simulated hypoxic conditions, a suitable way for mimicking the microenvironment of hypoxic solid tumour cells. AA interacted with the mitochondria and, upon photostimulation (10 min of light exposure), produced toxic amounts of reactive oxygen species (ROS), which damaged the organelle, as demonstrated by the high levels of lipid peroxidation and protein carbonylation. The photostimulated AA also depleted the GSH pool, which could compromise the mitochondrial antioxidant defence. Bioenergetically, AA photoinactivated the complexes I, II, and IV of the mitochondrial respiratory chain and the F1FO–ATP synthase complex, sharply inhibiting the oxidative phosphorylation. Upon photostimulation (10 min of light exposure), AA reduced the efficiency of mitochondrial energy transduction and oxidatively damaged lipids in isolated hepatocytes but did not decrease the viability of cells. Despite the useful photobiological properties, AA presented noticeable dark toxicity on mitochondrial bioenergetics, functioning predominantly as an uncoupler of oxidative phosphorylation. This harmful effect of AA was evidenced in isolated hepatocytes, in which AA diminished the cellular ATP content. In this case, the cells exhibited signs of cell viability reduction in the presence of high AA concentrations, but only after a long time of incubation (at least 90 min). The impairments on mitochondrial bioenergetics were also clearly manifested in intact perfused rat liver, in which AA diminished the cellular ATP content and stimulated the oxygen uptake. Consequently, gluconeogenesis and ureogenesis were strongly inhibited, whereas glycogenolysis and glycolysis were stimulated. AA also promoted the release of cytosolic and mitochondrial enzymes into the perfusate concomitantly with inhibition of oxygen consumption. In general, the intrinsic and photodynamic effects of AA were similar to those of MB, but AA caused some distinct effects such as the photoinactivation of the complex IV of the mitochondrial respiratory chain and a diminution of the ATP levels in the liver. It is evident that AA has the potential to be used in mitochondria-targeted photodynamic therapy, even under low oxygen concentrations. However, the fact that AA directly disrupts mitochondrial bioenergetics and affects several hepatic pathways that are linked to ATP metabolism, along with its ability to perturb cellular membranes and its little potential to reduce cell viability, could result in significant adverse effects especially in long-term treatments.