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Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes

Manika Das*, Sikha Bettina Mukherjee* and Chandrima Shaha{ddagger}

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
* These authors contributed equally to this work



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  		Fig. 1. Effects of H2O2 on the motility and viability of L. donovani promastigotes. (A) Percentage motility of cells after exposure to 0.1-8 mM doses of H2O2. (B) Percentage viability of cells after exposure to 0.1-8 mM doses of H2O2. Symbols: X, control without H2O2; +, 0.1 mM H2O2; m, 1 mM H2O2; ., 4 mM H2O2; r, 8 mM H2O2; d, 8 mM H2O2 + catalase; j, 4 mM H2O2 + catalase. The data represents mean ± s.e.m. of four experiments.

 


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  		Fig. 2. Cellular morphology, percent cells undergoing apoptosis-like death and nuclear morphology of L. donovani promastigotes after exposure to 4 mM H2O2. (A) Morphology. (a) Promastigotes not exposed to H2O2. (b) Promastigotes exposed to 4 mM H2O2 for 6 hours. (c) Promastigotes exposed to 4 mM H2O2 for 12 hours. Bar, 10 µm. (B) Percentage of cells undergoing apoptosis-like death as counted by a combination of Hoechst dye and PI staining. Symbols: j, control; m, 1 mM H2O2; ., 4 mM H2O2; d, 4 mM H2O2 + catalase (100 IU ml-1); r, 8 mM H2O2. (C) Changes in nuclei at different stages of cell death. Close ups of nuclei from a cell not exposed to H2O2 (a), a cell exposed to 4 mM H2O2 for 4 hours showing nuclear condensation (b) and a cell exposed to 4 mM H2O2 for 6 hours showing breakdown of nuclear material (c).

 


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  		Fig. 3. DNA fragmentation in L. donovani promastigotes with or without H2O2 treatment in culture as detected by TUNEL staining. (A) At 1 hour without H2O2 exposure. (B) At 1 hour with H2O2 treatment. (C) At 2 hours without H2O2 treatment. (D) At 2 hours after H2O2 treatment. (E) At 6 hours without H2O2 treatment. (F) At 6 hours after H2O2 treatment. (B1-F1) Scan-enhanced close ups of promastigotes with H2O2 treatment for 1 hour (B1), after 2 hours exposure to H2O2 (D1; notice the kinetoplast (k) staining) and after 6 hours exposure to H2O2 (F1; notice both kinetoplast and nuclei (n) staining). Bar, 10 µm.

 


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  		Fig. 4. DNA breakdown in L. donovani promastigotes after H2O2 exposure and effects of a caspase inhibitor on DNA breakage. (A) Number of cells positive for TUNEL staining at various hours after H2O2 treatment. Symbols: d, 4 mM H2O2 treatment; j, control with no treatment. Data are mean ± s.e.m. of four experiments. (B) DNA profile in agarose gels from treated and untreated promastigotes. (a) Without H2O2 exposure. (b) After 2 hours of 4 mM H2O2 treatment. (c) After 4 hours of 4 mM H2O2 exposure. (d) After 6 hours of 4 mM H2O2 treatment. (C) DNA breakage and the number of TdT labelled cells after inhibition of caspase activity by Z-DEVD-FMK. Symbols: j, 4 mM H2O2; m, 4 mM H2O2 + 1 µM inhibitor; ., 4 mM H2O2 + 10 µM inhibitor; d, control without treatment. Inset: DNA gel showing (a) marker, (b) DNA of cells pretreated with 1 µM inhibitor prior to exposure to 4 mM H2O2 and (c) DNA of cells treated with 4 mM H2O2. Data are mean ± s.e.m. of four experiments. (D) TdT labelled cells at 6 hours under different treatments. (a) Phase contrast of the same field as (b). (b) TdT labelled cells in 4 mM H2O2-treated group. (c) Phase contrast of same field as (d). (d). TdT labelled cells in group pretreated with Z-DEVD-FMK (1 µM) prior to exposure to H2O2. (e) Phase contrast of (f). (f) TdT labelled cells in the control group. Results are representative of three experiments. Bar, 100 µm.

 


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  		Fig. 5. Caspase activity and effects of inhibition of caspase activity. (A) Number of cells undergoing apoptosis-like death under different treatments. Symbols: j, treated with 4 mM H2O2; cells preincubated with CED-3/CPP32 group of protease inhibitor Z-DEVD-FMK (m, 1 µM; r, 10 µM; ., 25 µM) and 4 mM H2O2; d, control without any treatment. (B) Caspase activity in cell lysates with or without inhibitor treatment. Cell lysates were from L. donovani promastigotes at 30 minutes after exposure to 4 mM H2O2. Z-DEVD-FMK was used as a caspase-like protein inhibitor. Results are mean ± s.e.m. of three experiments. (C) Time course of PARP-like protein cleavage during H2O2-induced apoptosis in L. donovani promastigotes shown on western blots with anti-PARP antibody at different time points after H2O2 induced stress. Lanes: m, marker; 1, 0 hour; 2, 1 hour; 3, 2 hours; 4, 4 hours; 5, 6 hours. (D) PARP-like protein cleavage induced by H2O2 in the presence of a caspase inhibitor at 4 hours of treatment. Lanes: 1, with 4 mM H2O2 treatment; 2, control without treatment; 3, cells preincubated with 1 µg of Z-DEVD-FMK prior to exposure to 4 mM H2O2. Result is representative of three experiments.

 

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