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Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis.

Martínez-Espinosa R, Argüello-García R, Saavedra E, Ortega-Pierres G - Front Microbiol (2015)

Bottom Line: Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation.Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage.Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional México City, Mexico.

ABSTRACT
The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8, and 250 μM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a Giardia H2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

No MeSH data available.


Related in: MedlinePlus

Albendazole induces apoptosis-like death in Giardia. Annexin V binding in the parasite’s cell surface (as indicator of early apoptosis) was detected as a consequence of translocation of phosphatidylserine to the plasmatic membrane (middle panel). The parasites were exposed to DMF (A), 1.35 μM (B), 8 μM (C), 250 μM (D) of ABZ for 16 h at 37°C. Then annexin V-FITC and PI (indicator of late apoptosis and necrosis) were added to the medium. Trophozoites´ micrographs are as follows: top panel: in BF; bottom panel: trophozoites stained with annexin V-FITC and PI. The FITC and PI fluorescence was determined using a FACS Calibur Flow Cytometer. Data shown in the figure are as follows: quadrant R1 are cells positive for necrosis; quadrant R2 are cells positive for necrosis and apoptosis; quadrant R3 are cells negative for both markers; quadrant R4 are cells positive for early apoptosis. As control, flow cytometry was performed with WB cells exposed to 200 μM H2O2. The figures are representative of at least three independent experiments. The table shows population percent in each quadrant according to flow cytometry data.
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Figure 7: Albendazole induces apoptosis-like death in Giardia. Annexin V binding in the parasite’s cell surface (as indicator of early apoptosis) was detected as a consequence of translocation of phosphatidylserine to the plasmatic membrane (middle panel). The parasites were exposed to DMF (A), 1.35 μM (B), 8 μM (C), 250 μM (D) of ABZ for 16 h at 37°C. Then annexin V-FITC and PI (indicator of late apoptosis and necrosis) were added to the medium. Trophozoites´ micrographs are as follows: top panel: in BF; bottom panel: trophozoites stained with annexin V-FITC and PI. The FITC and PI fluorescence was determined using a FACS Calibur Flow Cytometer. Data shown in the figure are as follows: quadrant R1 are cells positive for necrosis; quadrant R2 are cells positive for necrosis and apoptosis; quadrant R3 are cells negative for both markers; quadrant R4 are cells positive for early apoptosis. As control, flow cytometry was performed with WB cells exposed to 200 μM H2O2. The figures are representative of at least three independent experiments. The table shows population percent in each quadrant according to flow cytometry data.

Mentions: To determine if ABZ causes necrosis or an apoptotic-like phenomenon, the translocation of phosphatidylserine in ABZ treated trophozoites was detected using anti-annexin V antibodies as a specific marker of early apoptosis. Trophozoites exposed to ABZ displayed a significant increase in annexin V staining. At higher drug concentration a significant number of cells positive to both markers indicated a process of cell death involving late apoptosis and necrosis (Figure 7). In further experiments the trophozoites nuclei were stained with PI to assess if ABZ exposure may alter the cell cycle progression between control and ABZ-exposed trophozoites. As can be observed in Figure 8, ABZ produced a noticeable and consistent decrease in the G1 subpopulation (yellow area, from 14.6% in vehicle-treated cells to 2.7% in 250 μM ABZ-treated cells), a slight decrease in the S-phase subpopulation in 1.35 μM ABZ-treated cells as compared to DMF-treated cells (stripped area, from 27 to 22.1%, respectively) and a moderated increase in G2 subpopulation between these same samples (blue area, from 61.0 to 69.9%, respectively). The cells treated with 8 μM ABZ displayed a similar distribution to the one observed in the population exposed to 250 μM ABZ. These data suggest that cytotoxic ABZ concentrations allow only a partial G1→S→G2 transit in cells in a pattern (G2 >> S > G1 > M) that indicates an arrested state at the G2/M phase boundary.


Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis.

Martínez-Espinosa R, Argüello-García R, Saavedra E, Ortega-Pierres G - Front Microbiol (2015)

Albendazole induces apoptosis-like death in Giardia. Annexin V binding in the parasite’s cell surface (as indicator of early apoptosis) was detected as a consequence of translocation of phosphatidylserine to the plasmatic membrane (middle panel). The parasites were exposed to DMF (A), 1.35 μM (B), 8 μM (C), 250 μM (D) of ABZ for 16 h at 37°C. Then annexin V-FITC and PI (indicator of late apoptosis and necrosis) were added to the medium. Trophozoites´ micrographs are as follows: top panel: in BF; bottom panel: trophozoites stained with annexin V-FITC and PI. The FITC and PI fluorescence was determined using a FACS Calibur Flow Cytometer. Data shown in the figure are as follows: quadrant R1 are cells positive for necrosis; quadrant R2 are cells positive for necrosis and apoptosis; quadrant R3 are cells negative for both markers; quadrant R4 are cells positive for early apoptosis. As control, flow cytometry was performed with WB cells exposed to 200 μM H2O2. The figures are representative of at least three independent experiments. The table shows population percent in each quadrant according to flow cytometry data.
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Figure 7: Albendazole induces apoptosis-like death in Giardia. Annexin V binding in the parasite’s cell surface (as indicator of early apoptosis) was detected as a consequence of translocation of phosphatidylserine to the plasmatic membrane (middle panel). The parasites were exposed to DMF (A), 1.35 μM (B), 8 μM (C), 250 μM (D) of ABZ for 16 h at 37°C. Then annexin V-FITC and PI (indicator of late apoptosis and necrosis) were added to the medium. Trophozoites´ micrographs are as follows: top panel: in BF; bottom panel: trophozoites stained with annexin V-FITC and PI. The FITC and PI fluorescence was determined using a FACS Calibur Flow Cytometer. Data shown in the figure are as follows: quadrant R1 are cells positive for necrosis; quadrant R2 are cells positive for necrosis and apoptosis; quadrant R3 are cells negative for both markers; quadrant R4 are cells positive for early apoptosis. As control, flow cytometry was performed with WB cells exposed to 200 μM H2O2. The figures are representative of at least three independent experiments. The table shows population percent in each quadrant according to flow cytometry data.
Mentions: To determine if ABZ causes necrosis or an apoptotic-like phenomenon, the translocation of phosphatidylserine in ABZ treated trophozoites was detected using anti-annexin V antibodies as a specific marker of early apoptosis. Trophozoites exposed to ABZ displayed a significant increase in annexin V staining. At higher drug concentration a significant number of cells positive to both markers indicated a process of cell death involving late apoptosis and necrosis (Figure 7). In further experiments the trophozoites nuclei were stained with PI to assess if ABZ exposure may alter the cell cycle progression between control and ABZ-exposed trophozoites. As can be observed in Figure 8, ABZ produced a noticeable and consistent decrease in the G1 subpopulation (yellow area, from 14.6% in vehicle-treated cells to 2.7% in 250 μM ABZ-treated cells), a slight decrease in the S-phase subpopulation in 1.35 μM ABZ-treated cells as compared to DMF-treated cells (stripped area, from 27 to 22.1%, respectively) and a moderated increase in G2 subpopulation between these same samples (blue area, from 61.0 to 69.9%, respectively). The cells treated with 8 μM ABZ displayed a similar distribution to the one observed in the population exposed to 250 μM ABZ. These data suggest that cytotoxic ABZ concentrations allow only a partial G1→S→G2 transit in cells in a pattern (G2 >> S > G1 > M) that indicates an arrested state at the G2/M phase boundary.

Bottom Line: Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation.Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage.Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional México City, Mexico.

ABSTRACT
The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8, and 250 μM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a Giardia H2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

No MeSH data available.


Related in: MedlinePlus