The photodynamic and direct actions of methylene blue on mitochondrial energy metabolism

Research performed at:

1. Department of Biochemistry, Laboratory of Biological Oxidations and Laboratory of Experimental Steatosis, Brazil

2. Department of Biochemistry, Laboratory of Plant Biochemistry, Maringá, Brazil

3. Department of Chemistry, State University of Maringá, Brazil.

The authors:

Eduardo Makiyama Klosowski, Byanca Thais Lima de Souza, Marcio Shigueaki Mito, Renato Polimeni Constantin, Gislaine Cristiane Mantovanelli, Juliana Morais Mewes, Paulo Francisco Veiga Bizerra, Paulo Vinicius Moreira da Costa Menezes, Eduardo Hideo Gilglioni, Karina Sayuri Utsunomiya, Rogério Marchiosi, Wanderley Dantas dos Santos, Osvaldo Ferrarese Filho, Wilker Caetano, Paulo Cesar de Souza Pereira, Renato Sonchini Gonçalves, Jorgete Constantin, Emy Luiza Ishii-Iwamoto, Rodrigo Polimeni Constantin.

According to the literature, methylene blue (MB) is a photosensitizer (PS) with a high affinity for mitochondria. Therefore, several studies have explored this feature to evaluate its photodynamic effects on the mitochondrial apoptotic pathway under normoxic conditions.

We are aware only of limited reports regarding MB’s photodynamic effects on mitochondrial energy metabolism, especially under hypoxic conditions. Thus, the purposes of this study were to determine the direct and photodynamic acute effects of MB on the energy metabolism of rat liver mitochondria under hypoxic conditions and its direct acute effects on several parameters linked to energy metabolism in the isolated perfused rat liver. MB presented a high affinity for mitochondria, irrespective of photostimulation or proton gradient formation.

Upon photostimulation, MB demonstrated high in vitro oxidizing species generation ability. Consequently, MB damaged the mitochondrial macromolecules, as could be evidenced by the elevated levels of lipid peroxidation and protein carbonyls. In addition to generating a prooxidant environment, MB also led to a deficient antioxidant defence system, as indicated by the reduced glutathione (GSH) depletion. Bioenergetically, MB caused uncoupling of oxidative phosphorylation and led to photodynamic inactivation of complex I, complex II, and F1F O–ATP synthase complex, thus decreasing mitochondrial ATP generation.

Contrary to what is expected for an ideal PS, MB displayed appreciable dark toxicity on mitochondrial energy metabolism. The results indicated that MB acted via at least three mechanisms: direct damage to the inner mitochondrial membrane; uncoupling of oxidative phosphorylation; and inhibition of electron transfer.

Confirming the impairment of mitochondrial energy metabolism, MB also strongly inhibited mitochondrial ATP production. In the perfused rat liver, MB stimulated oxygen consumption, decreased the ATP/ADP ratio, inhibited gluconeogenesis and ureogenesis, and stimulated glycogenolysis, glycolysis, and ammoniagenesis, fully corroborating its uncoupling action in intact cells, as well. It can be concluded that even under hypoxic conditions, MB is a PS with potential for photodynamic effect-induced mitochondrial dysfunction.