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

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.

Keywords: Giardia duodenalis; albendazole; antioxidant enzymes; drug resistance; sulfhydryl pool.

FIGURE 1

Differential levels of accumulation of ABZ and ABZ metabolites in Giardia duodenalis clones susceptible and resistant to ABZ. Trophozoites from ABZ-susceptible clone (WBDMF, open triangles) and a representative ABZ-resistant clone (WBR250, filled squares) were exposed to 10 μM ABZ for the times indicated in X-axis at 37°C and cell lysates were analyzed by HPLC using MBZ as internal standard. Chromatography assay was standardized using cell-free mixtures, adjusted to 13-min lasting elutions and each ABZ species was separated at the retention time indicated in the corresponding peak (A). These conditions were used in samples of cell lysates and the AUCs were used to determine the levels of ABZ (B), ABZ-SO (C), and ABZ-SOO (D). Results are the mean + SD. of three independent experiments and asterisks indicate statistical difference at P < 0.05.

FIGURE 2

Determination of ROS produced by ABZ exposure in ABZ-susceptible and ABZ-resistant clones. Trophozoites from the ABZ-susceptible clone WBDMF or the representative ABZ-resistant clone WBR250 were exposed to ABZ (test) or TBHP (positive control) at the concentrations indicated at the left for 24 h at 37°C and observed by light microscopy (LM) and cell fluorescence microscopy (ROS; A). The same samples were processed for flow cytometry analysis (B) with a window calibrated with fluorescein filters and readings of 20,000 events per sample. Graphs at the left are quadrant-based distributions of cells by size/granularity (X-axis) and fluorescence intensity (Y-axis) and histograms at the right show the quantitative displacement of cell fluorescence. Arrows indicate cells displaying profuse ROS staining. Scale bars: 20 μm.

FIGURE 3

Determination of NADHox and Pxr1a overexpression in ABZ-resistant G. duodenalis clones. Trophozoite lysates from clones susceptible (WBDMF) and resistant (WBR1.35, WBR8, and WBR250) to ABZ were separated by two dimensional gel electrophoresis (A) and the zones matching the expected molecular weight and isoelectric point of NADHox (dashed circle), FDP (circle), Pxr1a (dashed rectangle) and SOR (rectangle) were cut and processed by LC-MS/MS. Proteins detected and identified were NADHox (B) and Pxr1a (C) and the protein model with the scores of quality parameters is shown in the upper panel. The image analyses of protein spots in representative gels of each clone displaying differential peak volumes are shown in the lower panels. Ligands in protein models (ADP for NADHox and peroxide ion for Pxr1a) are shown in ball conformation.

FIGURE 4

Determination of mRNAs of antioxidant enzymes analyzed by PCR in ABZ-resistant G. duodenalis clones. Total RNA from trophozoites was reversely transcribed and the resulting cDNA was used to amplify by PCR four test loci that included pxr1a, nadhox, fdp and sor and two housekeeping loci (pdi1 and ubiq). Amplicons of expected size were identified by agarose gel electrophoresis in ABZ-susceptible and –resistant clones (A). Semi-quantitative densitometry using housekeeping loci to normalize data showed differential patterns of mRNA overexpression in ABZ-resistant clones for all antioxidant enzyme loci except sor (B). Data represent the mean values of three independent assays. M, size standards; control, PCR in absence of reverse transcriptase using RNA from the WBDMF clone.

FIGURE 5

Differential levels of the sulfhydryl pool in G. duodenalis trophozoites susceptible and resistant to ABZ. (A) Representative calibration curve using cysteine as standard including the corresponding linear regression coefficients. (B) Levels of free thiols determined in the ABZ-susceptible clone (open bar) and the ABZ-resistant clones (dark bars) using samples of 1.2 × 10⁷ trophozoites from each trophozoite culture. Data are the mean + SD. of three independent determinations. ^∗P < 0.05, ^∗∗P < 0.01.