Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway
Fig 5
L-arginine availability regulates redox balance in L. donovani promastigotes.
(A-E) Leishmania parasites were grown in L-arginine depleted (AD-Ld), L-arginine depleted but supplemented with ornithine (AD-Ld/Orn+), putrescine (AD-Ld/Put+), NAC (AD-Ld/NAC+) and L-arginine supplemented (AS-Ld) RPMI media for 0–120 hr. Intracellular ROS production was measured using the fluorescent dye 2, 7-dicholorodihydroflourescein diacetate (H2DCFDA) and NAC was used as a ROS scavenger (A). The rate of lipid peroxidation was measured by spectrofluorometry and expressed as fluorescence units at 430 nm (B). The total intracellular reduced thiol levels were measured spectrophotometrically using DTNB (C). SOD activity was measured by using SOD assay kit as described in “Materials and methods (D). The relative NADP+/NADPH ratio was measured spectrophotometrically based on the measurement of the absorbance of the reduced coenzyme at 450 nm (E). The data represents mean±SD of triplicate determinations and are representative of three independent experiments.*, P<0.05 (Student’s t test), **, P<0.001 compared to AD-Ld or AS-Ld parasite as applicable.