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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

DNA fragmentation and histone H2AX phosphorylation in G. duodenalis trophozoites exposed to ABZ. (A) WB strain trophozoites (WB) were exposed to 1.35, 8, and 250 μM of ABZ or to 100 μM of H2O2 in parallel with the resistance clones (R1.35, R.8, R.250) and the H2O2-resistant strain (ROX) which was exposed only to 8 μM of ABZ. Then the cells were incubated for 16 h at 37°C. Total DNA was extracted, treated with RNAse and analyzed in agarose gels. DNA degradation zone is shown by an arrow. DNA degradation was mainly observed in the WB strain treated with the different concentrations of ABZ (B). Histone H2AX phosphorylation was determined in total extract obtained from G. duodenalis WB trophozoites that were exposed to DMF and to different ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 16 h at 37°C. The reactivity of H2AX phosphorylation was determined by Western blot using rabbit anti-H2AX antibody. In the bottom of panel (B) the 15 kDa region of the Coomassie blue stained gel used as a load control is shown. The figures are representative of at least three independent experiments.
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Figure 6: DNA fragmentation and histone H2AX phosphorylation in G. duodenalis trophozoites exposed to ABZ. (A) WB strain trophozoites (WB) were exposed to 1.35, 8, and 250 μM of ABZ or to 100 μM of H2O2 in parallel with the resistance clones (R1.35, R.8, R.250) and the H2O2-resistant strain (ROX) which was exposed only to 8 μM of ABZ. Then the cells were incubated for 16 h at 37°C. Total DNA was extracted, treated with RNAse and analyzed in agarose gels. DNA degradation zone is shown by an arrow. DNA degradation was mainly observed in the WB strain treated with the different concentrations of ABZ (B). Histone H2AX phosphorylation was determined in total extract obtained from G. duodenalis WB trophozoites that were exposed to DMF and to different ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 16 h at 37°C. The reactivity of H2AX phosphorylation was determined by Western blot using rabbit anti-H2AX antibody. In the bottom of panel (B) the 15 kDa region of the Coomassie blue stained gel used as a load control is shown. The figures are representative of at least three independent experiments.

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)

DNA fragmentation and histone H2AX phosphorylation in G. duodenalis trophozoites exposed to ABZ. (A) WB strain trophozoites (WB) were exposed to 1.35, 8, and 250 μM of ABZ or to 100 μM of H2O2 in parallel with the resistance clones (R1.35, R.8, R.250) and the H2O2-resistant strain (ROX) which was exposed only to 8 μM of ABZ. Then the cells were incubated for 16 h at 37°C. Total DNA was extracted, treated with RNAse and analyzed in agarose gels. DNA degradation zone is shown by an arrow. DNA degradation was mainly observed in the WB strain treated with the different concentrations of ABZ (B). Histone H2AX phosphorylation was determined in total extract obtained from G. duodenalis WB trophozoites that were exposed to DMF and to different ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 16 h at 37°C. The reactivity of H2AX phosphorylation was determined by Western blot using rabbit anti-H2AX antibody. In the bottom of panel (B) the 15 kDa region of the Coomassie blue stained gel used as a load control is shown. The figures are representative of at least three independent experiments.
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Related In: Results  -  Collection

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

Figure 6: DNA fragmentation and histone H2AX phosphorylation in G. duodenalis trophozoites exposed to ABZ. (A) WB strain trophozoites (WB) were exposed to 1.35, 8, and 250 μM of ABZ or to 100 μM of H2O2 in parallel with the resistance clones (R1.35, R.8, R.250) and the H2O2-resistant strain (ROX) which was exposed only to 8 μM of ABZ. Then the cells were incubated for 16 h at 37°C. Total DNA was extracted, treated with RNAse and analyzed in agarose gels. DNA degradation zone is shown by an arrow. DNA degradation was mainly observed in the WB strain treated with the different concentrations of ABZ (B). Histone H2AX phosphorylation was determined in total extract obtained from G. duodenalis WB trophozoites that were exposed to DMF and to different ABZ concentrations (from left to right: 1.35, 8, and 250 μM) for 16 h at 37°C. The reactivity of H2AX phosphorylation was determined by Western blot using rabbit anti-H2AX antibody. In the bottom of panel (B) the 15 kDa region of the Coomassie blue stained gel used as a load control is shown. The figures are representative of at least three independent experiments.
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