An antioxidant response is involved in resistance of Giardia duodenalis to albendazole.

Albendazole (ABZ) is a therapeutic benzimidazole used to treat giardiasis that targets ß-tubulin. However, the molecular bases of ABZ resistance in Giardia duodenalis are not understood because ß-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.

Proteomic and transcriptional analyses of genes differentially expressed in Giardia duodenalis clones resistant to albendazole.

In this study we performed proteomic and transcriptional analyses to identify and characterize genes differentially expressed in Giardia duodenalis clones resistant to albendazole. The expression of proteins and their corresponding mRNAs was analyzed in clones resistant in vitro to different concentrations of albendazole (1.35, 8.0 and 250 µM) and these were compared with albendazole-sensitive clones using two approaches: (1) two-dimensional protein electrophoresis to analyze the proteome by the LC-MS/MS technique, and (2) semi-quantitative RT-PCR to assess the mRNA levels of proteins with the highest levels of differential expression .This strategy allowed the identification of eight proteins differentially expressed in albendazole resistant clones with roles in: (a) the cytoskeletal system (alpha 2-giardin and RanBP1), (b) the antioxidant metabolism (NADH oxidase) and (c) energy metabolism (triosephosphate isomerase, phosphoglycerate kinase and ornithine carbamoyltransferase). Gene expression analyses of these genes correlated well with the proteomics results. These observations suggest that resistance to albendazole in Giardia encompasses a complex response involving an altered expression of genes regulated at the transcriptional level that might have an important role in maintaining cell structural stability, coping with oxidative stress and adapting energy supply to a new metabolic status. These molecules are indeed promising targets for drug development.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro.

OBJECTIVES: We analysed, in a cell-by-cell study, the in vitro susceptibility of Giardia duodenalis strains, including Mexican isolates and their clones to 5-nitroimidazoles and benzimidazoles. METHODS: Fluorogenic dye staining (FDA-PI) and cell morphology (CM) assays, two fast and direct techniques, replaced the indirect 'gold standard' method (subculture in liquid medium) in the evaluation of 5-nitroimidazoles and benzimidazoles, respectively. RESULTS: Under these conditions, the activity of several 5-nitroimidazole and benzimidazole derivatives was consistent with their known efficacy, but parasite stocks exhibited a greater variability in response to 5-nitroimidazoles compared with benzimidazoles. Also, consecutive progenies from single stocks maintained in continuous subculture in drug-free media displayed changes (variations) in the proportions of drug resistant (R/T) subpopulations when exposed to sublethal concentrations of 5-nitroimidazoles and benzimidazoles. These were again more variable upon exposure to 5-nitroimidazoles than to benzimidazoles. Variations were not due to drug susceptibility shifts in parent trophozoites since analysis of cytokinetic processes showed a predominant pattern of susceptible/susceptible or resistant/resistant daughters, whereas susceptible/resistant daughters were scarce. CONCLUSIONS: Our observations support the idea that G. duodenalis cultures exhibit variations in their response to 5-nitroimidazoles and benzimidazoles as a result of a drug-independent competition between drug-susceptible and drug-resistant subpopulations when parasites are subcultured.