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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

Reactive oxygen species (ROS) are produced in Giardia duodenalis trophozoites exposed to Albendazole (ABZ). ABZ-sensitive Giardia strain (WB) was exposed to vehicle (DMF, A) and to 1.35 μM (B), 8 μM (C), or 250 μM (D) of ABZ for 8 h at 37°C, then DCFDH was added to monitor ROS production. In these experiments a control of intracellular ROS production was included. In this WB trophozoites were incubated with 100 μM H2O2(E). Trophozoites micrographs are as follows: top panels bright field (BF), middle panel trophozoites’ nuclei stained with Hoechst and lower panel trophozoites stained with DCFDH. The cells showed increased ROS production by ABZ treatment in comparison to control cells with no drug. Bottom panels are ROS production monitored by flow cytometry, the shift of fluorescence in X axis indicates ROS production by live trophozoites (determined by Trypan blue exclusion) at the highest ABZ concentration. The table shows population percent in M1 (negative to ROS) and M2 (positive to ROS) according to flow cytometry data. Micrographs are from representative results of at least three experiments performed with independent batch cultures.
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Figure 1: Reactive oxygen species (ROS) are produced in Giardia duodenalis trophozoites exposed to Albendazole (ABZ). ABZ-sensitive Giardia strain (WB) was exposed to vehicle (DMF, A) and to 1.35 μM (B), 8 μM (C), or 250 μM (D) of ABZ for 8 h at 37°C, then DCFDH was added to monitor ROS production. In these experiments a control of intracellular ROS production was included. In this WB trophozoites were incubated with 100 μM H2O2(E). Trophozoites micrographs are as follows: top panels bright field (BF), middle panel trophozoites’ nuclei stained with Hoechst and lower panel trophozoites stained with DCFDH. The cells showed increased ROS production by ABZ treatment in comparison to control cells with no drug. Bottom panels are ROS production monitored by flow cytometry, the shift of fluorescence in X axis indicates ROS production by live trophozoites (determined by Trypan blue exclusion) at the highest ABZ concentration. The table shows population percent in M1 (negative to ROS) and M2 (positive to ROS) according to flow cytometry data. Micrographs are from representative results of at least three experiments performed with independent batch cultures.

Mentions: In some reports using animal models ABZ was shown to produce oxidative damage (Locatelli et al., 2004; Bártíková et al., 2010). In G. duodenalis oxidative stress damage has been induced using pro-oxidant compounds such as H2O2 (Ghosh et al., 2009; Raj et al., 2013) in which intracellular ROS formation hallmarks this phenomenon. ABZ-exposed parasites showed greater ROS signals than parasites not exposed to the drug (Figure 1, top panels). The effect was mainly detected at the highest ABZ concentration tested (250 μM), however, ROS formation could be determined by flow cytometry at lower concentrations (Figure 1, bottom panels).


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)

Reactive oxygen species (ROS) are produced in Giardia duodenalis trophozoites exposed to Albendazole (ABZ). ABZ-sensitive Giardia strain (WB) was exposed to vehicle (DMF, A) and to 1.35 μM (B), 8 μM (C), or 250 μM (D) of ABZ for 8 h at 37°C, then DCFDH was added to monitor ROS production. In these experiments a control of intracellular ROS production was included. In this WB trophozoites were incubated with 100 μM H2O2(E). Trophozoites micrographs are as follows: top panels bright field (BF), middle panel trophozoites’ nuclei stained with Hoechst and lower panel trophozoites stained with DCFDH. The cells showed increased ROS production by ABZ treatment in comparison to control cells with no drug. Bottom panels are ROS production monitored by flow cytometry, the shift of fluorescence in X axis indicates ROS production by live trophozoites (determined by Trypan blue exclusion) at the highest ABZ concentration. The table shows population percent in M1 (negative to ROS) and M2 (positive to ROS) according to flow cytometry data. Micrographs are from representative results of at least three experiments performed with independent batch cultures.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Reactive oxygen species (ROS) are produced in Giardia duodenalis trophozoites exposed to Albendazole (ABZ). ABZ-sensitive Giardia strain (WB) was exposed to vehicle (DMF, A) and to 1.35 μM (B), 8 μM (C), or 250 μM (D) of ABZ for 8 h at 37°C, then DCFDH was added to monitor ROS production. In these experiments a control of intracellular ROS production was included. In this WB trophozoites were incubated with 100 μM H2O2(E). Trophozoites micrographs are as follows: top panels bright field (BF), middle panel trophozoites’ nuclei stained with Hoechst and lower panel trophozoites stained with DCFDH. The cells showed increased ROS production by ABZ treatment in comparison to control cells with no drug. Bottom panels are ROS production monitored by flow cytometry, the shift of fluorescence in X axis indicates ROS production by live trophozoites (determined by Trypan blue exclusion) at the highest ABZ concentration. The table shows population percent in M1 (negative to ROS) and M2 (positive to ROS) according to flow cytometry data. Micrographs are from representative results of at least three experiments performed with independent batch cultures.
Mentions: In some reports using animal models ABZ was shown to produce oxidative damage (Locatelli et al., 2004; Bártíková et al., 2010). In G. duodenalis oxidative stress damage has been induced using pro-oxidant compounds such as H2O2 (Ghosh et al., 2009; Raj et al., 2013) in which intracellular ROS formation hallmarks this phenomenon. ABZ-exposed parasites showed greater ROS signals than parasites not exposed to the drug (Figure 1, top panels). The effect was mainly detected at the highest ABZ concentration tested (250 μM), however, ROS formation could be determined by flow cytometry at lower concentrations (Figure 1, bottom panels).

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