Intracellular zinc depletion causes mitochondrial dysfunction in <i>L</i>. <i>donovani</i> promastigote.

<p><b>(PANEL I)</b> Zinc depleted and zinc supplemented <i>L</i>. <i>donovani</i> was cultivated in RPMI medium for indicated time periods and changes in mitochondrial membrane potential was determined by flow cytometry using TMRE dye. Plots are showing mean fluorescence intensity of TMRE positive cells after 16 hr (A) and 24 hr (B). FCCP was used as positive control for depolarization of mitochondrial membrane potential. <b>(PANEL II)</b> Zinc depleted and zinc supplemented <i>L</i>. <i>donovani</i> was cultivated in RPMI medium for indicated time periods and mitochondrial membrane depolarization was estimated by index of variation (IV) = (MT—MC)/MC, where MT corresponds to the median TMRE fluorescence of treated parasites and MC corresponds to the median TMRE fluorescence of control parasites (A). Intracellular calcium was measured by spectrofluorometry using Fura-2AM dye (B) and level of cytochrome c was checked in cytosolic fractions by western blotting (C) as described in “Materials & methods” section. EGTA was used as control for calcium chelation. Zinc depleted and zinc supplemented <i>L</i>. <i>donovani</i> was cultivated in RPMI medium for indicated time periods and caspase 3/7 activity was measured by spectrofluorometry as described in “Materials & methods” section. (D). DNA fragmentation was estimated by Cell death detection ELISA as described in materials and methods (E). The data represents Mean±S.D of three independent experiments in triplicates. * P< 0.05, **P< 0.001, *** P< 0.0001, ns Not significant; as compared in zinc depleted and zinc supplemented parasites with untreated control parasites by Kruskal-Wallis test with Dunn’s multiple comparison test.