Petiz 2013 Thesis Universidade Federal do Parana - Brazil

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Petiz LL (2013) Efeitos da N-N’-difenil-4-metoxi-benzamidina sobre parâmetros do metabolismo energético em mitocôndrias e hepatócitos. Thesis Universidade Federal do Parana - Brazil 91pp.

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Petiz LL (2013) Thesis Universidade Federal do Parana - Brazil

Abstract:

Keywords: Methoxyamidine; Mitochondrial bioenergetics; Hepatocytes; Toxicity


Labels: MiParea: Respiration, Pharmacology;toxicology 


Tissue;cell: Liver  Preparation: Intact cells, Isolated mitochondria 


Coupling state: LEAK, ROUTINE, OXPHOS  Pathway:HRR: Oxygraph-2k 


Amidines are chemically characterized by the presence of two nitrogen atoms bound to the same carbon atom in its structure. To these compounds several biological activities have been described. Pentamidine, an aromatic diamidine, is effective in the treatment of Pneumocystis carinii and leishmaniasis; however, this compound promotes severe side effects. For this reason, new amidine derivatives have been synthesized, among them the N-N’-diphenyl-4-methoxy-benzamidine (methoxyamidine), which was effective against L. amazonensis (LD50 20 μmol/L) and T. cruzi (LD50 59 nmol/L). In the present study, the toxicity of methoxyamidine was evaluated in mitochondria and isolated hepatocytes, wich was immediately used (suspension cells) or maintained in culture for 18 h. In mitochondria actively oxidizing glutamate and malate, the compound inhibited the state 3 respiration (25 nmol/mg of protein) by ~15% and the inhibition sites were the complex I and the segment between ubiquinone and complex III. Methoxyamidine also stimulated the state 4 respiration, at concentrations of 50 and 65 nmol/mg of protein by ~32% and ~43%, respectively. As a consequence of these effects, the respiratory control coefficient values (CCR) and the ADP/O ratio were decreased. The uncoupling effect of methoxyamidie, suggested by the stimulation of state 4 respiration, was confirmed by the dose dependent increase of oxygen consumption in state 4 induced by oligomycin, reaching to ~69% (65 nmol/mg of protein), and by the increased ATPase activity in intact mitochondria, by ~27% and ~64% to 50 and 65 nmol/mg of protein, respectively. Swelling supported by oxidation of glutamate plus malate in the presence of sodium acetate was inhibited by methoxyamidine by ~16 and 32% at concentrations of 50-65 nmol/mg of protein, respectively. Also the mitochondrial swelling in the absence of substrate and in the presence of K+ and valinomycin, was inhibited by ~20% at the same concentrations, suggesting that methoxyamidine affects the mitochondrial membrane permeability and fluidity. In isolated hepatocytes, the effects of methoxyamidine and pentamidine were compared, at concentrations of 25 and 65 μmol/L. In the respiration assays with suspension cells, the results of methoxyamidine were compatible with those obtained in isolated mitochondria, with a decrease in basal (~8% - 65 μmol/L) and uncoupled (~26% - 65 μmol/L) respiration states. Leak state was stimulated by ~47% by methoxyamidine (65 μmol/L). For pentamidine the results were similar. Basal and uncoupled cellular respiration was inhibited at ~18% by pentamidine (65 μmol/L). In suspension cells, pyruvate levels were not affected by the compounds whereas lactate levels were increased by ~17% in the highest concentration (65 μmol/L) of methoxyamidine after 40min of incubation. In hepatocytes maintained in culture, the methoxyamidine treatment (65 μmol/L – 18 h) increased the cell viability by ~68% and ~60%, evaluated by MTT and LDH activity, respectively. Pentamidine (65 μmol/L – 18 h) decreased in ~68% and 258% the hepatocyte viability, evaluated by MTT and LDH activity, respectively. Taken together these results show that methoxyamidine at concentration similar to LD50 (20 μmol/L) for L. amazonensis, promotes discreet effects on mitochondrial bioenergetics. These effects were also observed in suspension hepatocytes; however, when these cells were maintained in culture, the compound seemed to exert a citoprotector effect.