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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

Oxidative damage to DNA in G. duodenalis trophozoites exposed to ABZ. Parasites of the WB strain were exposed to DMF, 1.35 μM, 8 μM, 250 μM of ABZ or 100 μM of H2O2 (the latter as control of ROS damage) for 8 h at 37°C. Then trophozoites were fixed on slides and incubated with the anti-8-hydroxydeoxyguanosine monoclonal antibody to detect modified nucleotides in DNA as described in materials and methods. Trophozoite micrographs are as follows: top panel trophozoites’ nuclei stained with Hoechst; lower panel reactivity of anti-8-hydroxydeoxyguanosine (8OHdG) antibodies in trophozoites’ nuclei (WB + 8 μM ABZ) and cytoplasm (WB + 250 μM ABZ). The figures are representative of at least three independent experiments.
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Figure 5: Oxidative damage to DNA in G. duodenalis trophozoites exposed to ABZ. Parasites of the WB strain were exposed to DMF, 1.35 μM, 8 μM, 250 μM of ABZ or 100 μM of H2O2 (the latter as control of ROS damage) for 8 h at 37°C. Then trophozoites were fixed on slides and incubated with the anti-8-hydroxydeoxyguanosine monoclonal antibody to detect modified nucleotides in DNA as described in materials and methods. Trophozoite micrographs are as follows: top panel trophozoites’ nuclei stained with Hoechst; lower panel reactivity of anti-8-hydroxydeoxyguanosine (8OHdG) antibodies in trophozoites’ nuclei (WB + 8 μM ABZ) and cytoplasm (WB + 250 μM ABZ). The figures are representative of at least three independent experiments.

Mentions: Oxidative damage to DNA by ABZ treatment was determined by immunocytochemistry assays using antibodies against 8-OHdG. In these, a significant increase in intranuclear staining, denoting 8-OHdG-containing DNA in ABZ-treated cells was observed (Figure 5). At the highest drug concentrations used (250 μM) the fluorescent signal was observed in nucleus and the entire cytosol displayed a diffuse pattern with a punctuated pattern in some regions within the trophozoites (Figure 5).


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)

Oxidative damage to DNA in G. duodenalis trophozoites exposed to ABZ. Parasites of the WB strain were exposed to DMF, 1.35 μM, 8 μM, 250 μM of ABZ or 100 μM of H2O2 (the latter as control of ROS damage) for 8 h at 37°C. Then trophozoites were fixed on slides and incubated with the anti-8-hydroxydeoxyguanosine monoclonal antibody to detect modified nucleotides in DNA as described in materials and methods. Trophozoite micrographs are as follows: top panel trophozoites’ nuclei stained with Hoechst; lower panel reactivity of anti-8-hydroxydeoxyguanosine (8OHdG) antibodies in trophozoites’ nuclei (WB + 8 μM ABZ) and cytoplasm (WB + 250 μM ABZ). The figures 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 5: Oxidative damage to DNA in G. duodenalis trophozoites exposed to ABZ. Parasites of the WB strain were exposed to DMF, 1.35 μM, 8 μM, 250 μM of ABZ or 100 μM of H2O2 (the latter as control of ROS damage) for 8 h at 37°C. Then trophozoites were fixed on slides and incubated with the anti-8-hydroxydeoxyguanosine monoclonal antibody to detect modified nucleotides in DNA as described in materials and methods. Trophozoite micrographs are as follows: top panel trophozoites’ nuclei stained with Hoechst; lower panel reactivity of anti-8-hydroxydeoxyguanosine (8OHdG) antibodies in trophozoites’ nuclei (WB + 8 μM ABZ) and cytoplasm (WB + 250 μM ABZ). The figures are representative of at least three independent experiments.
Mentions: Oxidative damage to DNA by ABZ treatment was determined by immunocytochemistry assays using antibodies against 8-OHdG. In these, a significant increase in intranuclear staining, denoting 8-OHdG-containing DNA in ABZ-treated cells was observed (Figure 5). At the highest drug concentrations used (250 μM) the fluorescent signal was observed in nucleus and the entire cytosol displayed a diffuse pattern with a punctuated pattern in some regions within the trophozoites (Figure 5).

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