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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

Cross-resistance to ABZ and H2O2 and cysteine (Cys) protection in trophozoites exposed to ABZ. (A) Cross-resistance to H2O2 was evaluated in the ABZ-resistant clones R1.35 (black bars), R.8 (horizontal lined bars), and R.250 (vertical lined bars) previously obtained in our group (Paz-Maldonado et al., 2013). Parasites were exposed to the indicated H2O2 concentrations for 24 h and the cell number was determined by SYTOX Green. As control, wild type WB strain (white bars) was incubated with the indicated H2O2 concentration. (B) Cross-resistance to ABZ in a H2O2-resistant G. dudodenalis. A H2O2-resistant Giardia strain (ROX) was obtained by sub-culturing the WB strain in increasing H2O2 concentration for 6 months. Control WB (white bars) and ROX (gray bars) trophozoites were exposed to the indicated increasing ABZ concentrations for 24 h and the cell number was determined using the fluorescent tracer SYTOX Green. The ROX strain showed cross-resistance to ABZ. (C) Cysteine protection of Giardia trophozoites exposed to ABZ. WB trophozoites were incubated in growth medium with 0.5 mM (white bar), 1 mM (lined up to the right bar), 2 mM (lined up to the left bar), or 4 mM (boxed bar) of cysteine for 24 h and then trophozoites were further incubated with 0.2 μM ABZ for 48 h. In all graphs the results are the mean ± SD of at least three independent experiments. In graphs (B,C)∗ indicates p ≤ 0.05 by ANOVA and Tukey´s analysis in which values obtained with WB trophozoites were compared with values obtained in ROX trophozoites exposed to the different ABZ concentrations (B). (C) Values obtained in WB trophozoites treated with 0.5 mM cysteine were compared to values obtained in WB trophozoites treated with different cysteine concentrations and then exposed to ABZ.
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Figure 3: Cross-resistance to ABZ and H2O2 and cysteine (Cys) protection in trophozoites exposed to ABZ. (A) Cross-resistance to H2O2 was evaluated in the ABZ-resistant clones R1.35 (black bars), R.8 (horizontal lined bars), and R.250 (vertical lined bars) previously obtained in our group (Paz-Maldonado et al., 2013). Parasites were exposed to the indicated H2O2 concentrations for 24 h and the cell number was determined by SYTOX Green. As control, wild type WB strain (white bars) was incubated with the indicated H2O2 concentration. (B) Cross-resistance to ABZ in a H2O2-resistant G. dudodenalis. A H2O2-resistant Giardia strain (ROX) was obtained by sub-culturing the WB strain in increasing H2O2 concentration for 6 months. Control WB (white bars) and ROX (gray bars) trophozoites were exposed to the indicated increasing ABZ concentrations for 24 h and the cell number was determined using the fluorescent tracer SYTOX Green. The ROX strain showed cross-resistance to ABZ. (C) Cysteine protection of Giardia trophozoites exposed to ABZ. WB trophozoites were incubated in growth medium with 0.5 mM (white bar), 1 mM (lined up to the right bar), 2 mM (lined up to the left bar), or 4 mM (boxed bar) of cysteine for 24 h and then trophozoites were further incubated with 0.2 μM ABZ for 48 h. In all graphs the results are the mean ± SD of at least three independent experiments. In graphs (B,C)∗ indicates p ≤ 0.05 by ANOVA and Tukey´s analysis in which values obtained with WB trophozoites were compared with values obtained in ROX trophozoites exposed to the different ABZ concentrations (B). (C) Values obtained in WB trophozoites treated with 0.5 mM cysteine were compared to values obtained in WB trophozoites treated with different cysteine concentrations and then exposed to ABZ.

Mentions: The ABZ-resistant clones, namely R1.35, R.8, and R.250 (Argüello-García et al., 2009; Paz-Maldonado et al., 2013) were used to determine whether cross-resistance to classical oxidative stressor (H2O2) and ABZ was induced in the resistant trophozoites. For this purpose the ABZ-resistant clones were incubated under increasing concentrations of H2O2, and cell growth was determined. In general the resistant clones R1.35 and R.250 showed a tendency to increased resistance to H2O2-induced death in comparison to the ABZ-susceptible WB strain (Figure 3A). A special case is the R8 resistant strain which frequently behave, in this and other studies, as a “transition state” between low and high ABZ resistance depending on the parameter that is evaluated (see also Figure 6A; Argüello-García et al., 2009; Paz-Maldonado et al., 2013)


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)

Cross-resistance to ABZ and H2O2 and cysteine (Cys) protection in trophozoites exposed to ABZ. (A) Cross-resistance to H2O2 was evaluated in the ABZ-resistant clones R1.35 (black bars), R.8 (horizontal lined bars), and R.250 (vertical lined bars) previously obtained in our group (Paz-Maldonado et al., 2013). Parasites were exposed to the indicated H2O2 concentrations for 24 h and the cell number was determined by SYTOX Green. As control, wild type WB strain (white bars) was incubated with the indicated H2O2 concentration. (B) Cross-resistance to ABZ in a H2O2-resistant G. dudodenalis. A H2O2-resistant Giardia strain (ROX) was obtained by sub-culturing the WB strain in increasing H2O2 concentration for 6 months. Control WB (white bars) and ROX (gray bars) trophozoites were exposed to the indicated increasing ABZ concentrations for 24 h and the cell number was determined using the fluorescent tracer SYTOX Green. The ROX strain showed cross-resistance to ABZ. (C) Cysteine protection of Giardia trophozoites exposed to ABZ. WB trophozoites were incubated in growth medium with 0.5 mM (white bar), 1 mM (lined up to the right bar), 2 mM (lined up to the left bar), or 4 mM (boxed bar) of cysteine for 24 h and then trophozoites were further incubated with 0.2 μM ABZ for 48 h. In all graphs the results are the mean ± SD of at least three independent experiments. In graphs (B,C)∗ indicates p ≤ 0.05 by ANOVA and Tukey´s analysis in which values obtained with WB trophozoites were compared with values obtained in ROX trophozoites exposed to the different ABZ concentrations (B). (C) Values obtained in WB trophozoites treated with 0.5 mM cysteine were compared to values obtained in WB trophozoites treated with different cysteine concentrations and then exposed to ABZ.
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Related In: Results  -  Collection

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

Figure 3: Cross-resistance to ABZ and H2O2 and cysteine (Cys) protection in trophozoites exposed to ABZ. (A) Cross-resistance to H2O2 was evaluated in the ABZ-resistant clones R1.35 (black bars), R.8 (horizontal lined bars), and R.250 (vertical lined bars) previously obtained in our group (Paz-Maldonado et al., 2013). Parasites were exposed to the indicated H2O2 concentrations for 24 h and the cell number was determined by SYTOX Green. As control, wild type WB strain (white bars) was incubated with the indicated H2O2 concentration. (B) Cross-resistance to ABZ in a H2O2-resistant G. dudodenalis. A H2O2-resistant Giardia strain (ROX) was obtained by sub-culturing the WB strain in increasing H2O2 concentration for 6 months. Control WB (white bars) and ROX (gray bars) trophozoites were exposed to the indicated increasing ABZ concentrations for 24 h and the cell number was determined using the fluorescent tracer SYTOX Green. The ROX strain showed cross-resistance to ABZ. (C) Cysteine protection of Giardia trophozoites exposed to ABZ. WB trophozoites were incubated in growth medium with 0.5 mM (white bar), 1 mM (lined up to the right bar), 2 mM (lined up to the left bar), or 4 mM (boxed bar) of cysteine for 24 h and then trophozoites were further incubated with 0.2 μM ABZ for 48 h. In all graphs the results are the mean ± SD of at least three independent experiments. In graphs (B,C)∗ indicates p ≤ 0.05 by ANOVA and Tukey´s analysis in which values obtained with WB trophozoites were compared with values obtained in ROX trophozoites exposed to the different ABZ concentrations (B). (C) Values obtained in WB trophozoites treated with 0.5 mM cysteine were compared to values obtained in WB trophozoites treated with different cysteine concentrations and then exposed to ABZ.
Mentions: The ABZ-resistant clones, namely R1.35, R.8, and R.250 (Argüello-García et al., 2009; Paz-Maldonado et al., 2013) were used to determine whether cross-resistance to classical oxidative stressor (H2O2) and ABZ was induced in the resistant trophozoites. For this purpose the ABZ-resistant clones were incubated under increasing concentrations of H2O2, and cell growth was determined. In general the resistant clones R1.35 and R.250 showed a tendency to increased resistance to H2O2-induced death in comparison to the ABZ-susceptible WB strain (Figure 3A). A special case is the R8 resistant strain which frequently behave, in this and other studies, as a “transition state” between low and high ABZ resistance depending on the parameter that is evaluated (see also Figure 6A; Argüello-García et al., 2009; Paz-Maldonado et al., 2013)

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