Limits...
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

Intracellular localization of ROS production in G. dudodenalis trophozoites exposed to ABZ. WB Giardia trophozoites were exposed to DMF or to the indicated ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 8 h at 37°C. Cells were then incubated with DCFDH. Trophozoite micrographs are as follows: (A) top panel: representative images of trophozoites exposed to DMF (a,b) or to 250 μM ABZ (c,d) and then incubated with DCFDH. Morphological changes in trophozoites (c BF) and ROS localization (d epifluorescence illumination) are evident in ABZ treated cells. (B) Images of representative individual cells. Top panel trophozoites´ nuclei stained with Hoechst, middle panel trophozoites incubated with DCFDH (epifluorescence illumination) and lower panel merged cell images. At the lowest concentrations, ROS production is restricted to nuclei, whereas at the highest ABZ concentration, this is detected all over the cytoplasm. The micrographs are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526806&req=5

Figure 2: Intracellular localization of ROS production in G. dudodenalis trophozoites exposed to ABZ. WB Giardia trophozoites were exposed to DMF or to the indicated ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 8 h at 37°C. Cells were then incubated with DCFDH. Trophozoite micrographs are as follows: (A) top panel: representative images of trophozoites exposed to DMF (a,b) or to 250 μM ABZ (c,d) and then incubated with DCFDH. Morphological changes in trophozoites (c BF) and ROS localization (d epifluorescence illumination) are evident in ABZ treated cells. (B) Images of representative individual cells. Top panel trophozoites´ nuclei stained with Hoechst, middle panel trophozoites incubated with DCFDH (epifluorescence illumination) and lower panel merged cell images. At the lowest concentrations, ROS production is restricted to nuclei, whereas at the highest ABZ concentration, this is detected all over the cytoplasm. The micrographs are representative of at least three independent experiments.

Mentions: To determine the localization of intracellular ROS formation within the trophozoites confocal microscopy was used. In these experiments the typical altered morphology caused by benzimidazoles (Paz-Maldonado et al., 2013) was observed in ABZ-treated trophozoites (Figure 2A top panel c). In these cells ROS formation was also evident in most cells as determined by fluorescent staining (Figure 2A top panel d). When individual cells were observed the trophozoites´nuclei were determined as the primary site of ROS formation (Figure 2B) as judged by fluorescent staining at low ABZ concentrations used (1.35 μM and 8 μM). At the highest drug concentration used (250 μM) there was a widespread distribution of ROS throughout the trophozoite cytoplasm (Figure 2B middle panel).


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)

Intracellular localization of ROS production in G. dudodenalis trophozoites exposed to ABZ. WB Giardia trophozoites were exposed to DMF or to the indicated ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 8 h at 37°C. Cells were then incubated with DCFDH. Trophozoite micrographs are as follows: (A) top panel: representative images of trophozoites exposed to DMF (a,b) or to 250 μM ABZ (c,d) and then incubated with DCFDH. Morphological changes in trophozoites (c BF) and ROS localization (d epifluorescence illumination) are evident in ABZ treated cells. (B) Images of representative individual cells. Top panel trophozoites´ nuclei stained with Hoechst, middle panel trophozoites incubated with DCFDH (epifluorescence illumination) and lower panel merged cell images. At the lowest concentrations, ROS production is restricted to nuclei, whereas at the highest ABZ concentration, this is detected all over the cytoplasm. The micrographs are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526806&req=5

Figure 2: Intracellular localization of ROS production in G. dudodenalis trophozoites exposed to ABZ. WB Giardia trophozoites were exposed to DMF or to the indicated ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 8 h at 37°C. Cells were then incubated with DCFDH. Trophozoite micrographs are as follows: (A) top panel: representative images of trophozoites exposed to DMF (a,b) or to 250 μM ABZ (c,d) and then incubated with DCFDH. Morphological changes in trophozoites (c BF) and ROS localization (d epifluorescence illumination) are evident in ABZ treated cells. (B) Images of representative individual cells. Top panel trophozoites´ nuclei stained with Hoechst, middle panel trophozoites incubated with DCFDH (epifluorescence illumination) and lower panel merged cell images. At the lowest concentrations, ROS production is restricted to nuclei, whereas at the highest ABZ concentration, this is detected all over the cytoplasm. The micrographs are representative of at least three independent experiments.
Mentions: To determine the localization of intracellular ROS formation within the trophozoites confocal microscopy was used. In these experiments the typical altered morphology caused by benzimidazoles (Paz-Maldonado et al., 2013) was observed in ABZ-treated trophozoites (Figure 2A top panel c). In these cells ROS formation was also evident in most cells as determined by fluorescent staining (Figure 2A top panel d). When individual cells were observed the trophozoites´nuclei were determined as the primary site of ROS formation (Figure 2B) as judged by fluorescent staining at low ABZ concentrations used (1.35 μM and 8 μM). At the highest drug concentration used (250 μM) there was a widespread distribution of ROS throughout the trophozoite cytoplasm (Figure 2B middle panel).

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