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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 proteins in G. duodenalis trophozoites exposed to ABZ. (A) Protein carbonylation in G. duodenalis ABZ-sensitive trophozoites (WB) exposed to ABZ. Parasites were exposed to vehicle (DMF white bars) or to different ABZ concentrations (1.35 μM black bars; 8 μM horizontal lined bars; 250 μM vertical lined bars) for 16 h at 37°C. Control WB trophozoites were exposed to direct ROS damage with 100 μM H2O2 (white and black boxes). Proteins from each sample were obtained and were derivatized with DNPH for protein cabonylation determination. DNP-protein adducts were detected by absorbance at 450 nm. (B) Lipoperoxidation in trophozoites exposed to ABZ. After trophozoites were exposed to the different ABZ concentrations as indicated in (A). HNE or MDA were determined by absorbance at 586 nm. The values in (A,B) are the mean ± SD. ∗p ≤ 0.05 by ANOVA and Tukey’s analysis in which values obtained with trophozoites treated with the different ABZ concentrations were compared to values obtained with the DMF treated parasites. (C) MDA-protein adducts were detected by Western blot in the same trophozoites samples treated with the indicated ABZ concentrations using rabbit anti-MDA antibodies. At the bottom of panel (C) the Coomassie blue stained gel is included and shows that similar protein amounts of each sample were loaded.
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Figure 4: Oxidative damage to proteins in G. duodenalis trophozoites exposed to ABZ. (A) Protein carbonylation in G. duodenalis ABZ-sensitive trophozoites (WB) exposed to ABZ. Parasites were exposed to vehicle (DMF white bars) or to different ABZ concentrations (1.35 μM black bars; 8 μM horizontal lined bars; 250 μM vertical lined bars) for 16 h at 37°C. Control WB trophozoites were exposed to direct ROS damage with 100 μM H2O2 (white and black boxes). Proteins from each sample were obtained and were derivatized with DNPH for protein cabonylation determination. DNP-protein adducts were detected by absorbance at 450 nm. (B) Lipoperoxidation in trophozoites exposed to ABZ. After trophozoites were exposed to the different ABZ concentrations as indicated in (A). HNE or MDA were determined by absorbance at 586 nm. The values in (A,B) are the mean ± SD. ∗p ≤ 0.05 by ANOVA and Tukey’s analysis in which values obtained with trophozoites treated with the different ABZ concentrations were compared to values obtained with the DMF treated parasites. (C) MDA-protein adducts were detected by Western blot in the same trophozoites samples treated with the indicated ABZ concentrations using rabbit anti-MDA antibodies. At the bottom of panel (C) the Coomassie blue stained gel is included and shows that similar protein amounts of each sample were loaded.

Mentions: The formation of intracellular ROS may trigger oxidative damage to various biomolecules such as proteins, lipids, and DNA. Protein carbonylation was determined in ABZ-sensitive trophozoites exposed to increasing ABZ concentrations. As can be seen in Figure 4A, a consistent but not statistically significant increase in protein carbonylation correlated with the increase in drug concentration, confirming the presence of oxidative damage by ABZ treatment in cellular proteins of G. duodenalis.


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 proteins in G. duodenalis trophozoites exposed to ABZ. (A) Protein carbonylation in G. duodenalis ABZ-sensitive trophozoites (WB) exposed to ABZ. Parasites were exposed to vehicle (DMF white bars) or to different ABZ concentrations (1.35 μM black bars; 8 μM horizontal lined bars; 250 μM vertical lined bars) for 16 h at 37°C. Control WB trophozoites were exposed to direct ROS damage with 100 μM H2O2 (white and black boxes). Proteins from each sample were obtained and were derivatized with DNPH for protein cabonylation determination. DNP-protein adducts were detected by absorbance at 450 nm. (B) Lipoperoxidation in trophozoites exposed to ABZ. After trophozoites were exposed to the different ABZ concentrations as indicated in (A). HNE or MDA were determined by absorbance at 586 nm. The values in (A,B) are the mean ± SD. ∗p ≤ 0.05 by ANOVA and Tukey’s analysis in which values obtained with trophozoites treated with the different ABZ concentrations were compared to values obtained with the DMF treated parasites. (C) MDA-protein adducts were detected by Western blot in the same trophozoites samples treated with the indicated ABZ concentrations using rabbit anti-MDA antibodies. At the bottom of panel (C) the Coomassie blue stained gel is included and shows that similar protein amounts of each sample were loaded.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526806&req=5

Figure 4: Oxidative damage to proteins in G. duodenalis trophozoites exposed to ABZ. (A) Protein carbonylation in G. duodenalis ABZ-sensitive trophozoites (WB) exposed to ABZ. Parasites were exposed to vehicle (DMF white bars) or to different ABZ concentrations (1.35 μM black bars; 8 μM horizontal lined bars; 250 μM vertical lined bars) for 16 h at 37°C. Control WB trophozoites were exposed to direct ROS damage with 100 μM H2O2 (white and black boxes). Proteins from each sample were obtained and were derivatized with DNPH for protein cabonylation determination. DNP-protein adducts were detected by absorbance at 450 nm. (B) Lipoperoxidation in trophozoites exposed to ABZ. After trophozoites were exposed to the different ABZ concentrations as indicated in (A). HNE or MDA were determined by absorbance at 586 nm. The values in (A,B) are the mean ± SD. ∗p ≤ 0.05 by ANOVA and Tukey’s analysis in which values obtained with trophozoites treated with the different ABZ concentrations were compared to values obtained with the DMF treated parasites. (C) MDA-protein adducts were detected by Western blot in the same trophozoites samples treated with the indicated ABZ concentrations using rabbit anti-MDA antibodies. At the bottom of panel (C) the Coomassie blue stained gel is included and shows that similar protein amounts of each sample were loaded.
Mentions: The formation of intracellular ROS may trigger oxidative damage to various biomolecules such as proteins, lipids, and DNA. Protein carbonylation was determined in ABZ-sensitive trophozoites exposed to increasing ABZ concentrations. As can be seen in Figure 4A, a consistent but not statistically significant increase in protein carbonylation correlated with the increase in drug concentration, confirming the presence of oxidative damage by ABZ treatment in cellular proteins of G. duodenalis.

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