Abstract
Albendazole (ABZ) is a therapeutic benzimidazole used to treat giardiasis that targets β-tubulin. However, the molecular bases of ABZ resistance in Giardia duodenalis are not understood because β-tubulin in ABZ-resistant clones lacks mutations explaining drug resistance. In previous work we compared ABZ-resistant (1.35, 8, and 250 μM) and ABZ-susceptible clones by proteomic analysis and eight proteins involved in energy metabolism, cytoskeleton dynamics, and antioxidant response were found as differentially expressed among the clones. Since ABZ is converted into sulphoxide (ABZ-SO) and sulphone (ABZ-SOO) metabolites we measured the levels of these metabolites, the antioxidant enzymes and free thiols in the susceptible and resistant clones. Production of reactive oxygen species (ROS) and levels of ABZ-SO/ABZ-SOO induced by ABZ were determined by fluorescein diacetate-based fluorescence and liquid chromatography respectively. The mRNA and protein levels of antioxidant enzymes (NADH oxidase, peroxiredoxin 1a, superoxide dismutase and flavodiiron protein) in these clones were determined by RT-PCR and proteomic analysis. The intracellular sulfhydryl (R-SH) pool was quantified using dinitrobenzoic acid. The results showed that ABZ induced ROS accumulation in the ABZ-susceptible Giardia cultures but not in the resistant ones whilst the accumulation of ABZ-SO and ABZ-SOO was lower in all ABZ-resistant cultures. Consistent with these findings, all the antioxidant enzymes detected and analyzed were upregulated in ABZ-resistant clones. Likewise the R-SH pool increased concomitantly to the degree of ABZ-resistance. These results indicate an association between accumulation of ABZ metabolites and a pro-oxidant effect of ABZ in Giardia-susceptible clones. Furthermore the antioxidant response involving ROS-metabolizing enzymes and intracellular free thiols in ABZ-resistant parasites suggest that this response may contribute to overcome the pro-oxidant cytotoxicity of ABZ.
Highlights
ABZ Metabolites are Differentially Accumulated in G. duodenalis Clones Susceptible and Resistant to ABZ In this study, we initially determined if ABZ is metabolized, i.e., oxidized by G. duodenalis trophozoites
The increasing baseline starting at 6-10 min of elution, peaks at 10 min and drops to initial A292 values by 12 min (Figure 1A) was due to the changing proportions of acetonitrile in the mobile phase and not by artifacts from any of tested compounds, especially ABZ and MBZ for which area under curve (AUC) were obtained considering these changes in baselines
MRNAs of Several Antioxidant Enzymes are Upregulated in ABZ-resistant G. duodenalis Trophozoites
Summary
G. lamblia, G. intestinalis) is the protozoan causing giardiasis that is a leading cause of parasitic diarrheal disease in humans and animals. According to estimations of the World Health Organization (WHO), giardiasis accounts for almost a billion cases worldwide with ∼3 billion people living in areas in which the incidence of the infection is around 30% (Escobedo et al, 2010). The socioeconomic and clinical impact of giardiasis has prompted its inclusion within the WHO’s Neglected Diseases Initiative (Savioli et al, 2006). The control of this infection requires both the inactivation of the infectious cysts disseminated in the environment and the elimination of pathogenic trophozoites that attach to small intestinal epithelium. Adult asymptomatic carriers are frequently observed (Adam, 2001; Escobedo and Cimerman, 2007)
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