INTRODUCTION
The trypanosomatids, members of the order Kinetoplastida include parasitic protozoa such as Leishmania spp. that are responsible for Leishmaniasis (Herwaldt, Reference Herwaldt1999; Guerin et al. Reference Guerin, Olliaro, Sundar, Boelaert, Croft, Desjeux, Wasunna and Bryceson2002). The disease endemicity extends to over 88 countries, 72 being in the developing world while 13 belong to the category of least developed countries (Murray et al. Reference Murray, Berman, Davies and Saravia2005). Globally, the population at risk is 350 million, overall prevalence being 12 million, 2 million new cases occur annually and the Disability adjusted Life years (DALY) burden is considered to be 860 000 men and 1·2 million women (TDR, 2005). The estimated annual incidence of the visceral form is around 500 000 in 61 countries with 90% of these cases being confined to 5 countries namely India (especially the state of Bihar and its adjoining states), Bangladesh, Nepal (Terai region), Sudan and North Eastern Brazil (Guerin et al. Reference Guerin, Olliaro, Sundar, Boelaert, Croft, Desjeux, Wasunna and Bryceson2002).
An increasing incidence of unresponsiveness to sodium antimony gluconate (SAG) is a critical issue in the current, prolonged epidemic in India where over 50–65% of the estimated 250 000 annual cases are non-responsive to this first-line agent (Croft et al. Reference Croft, Sundar and Fairlamb2006). Mechanism(s) by which Leishmania spp. acquire resistance to antimony is a subject of intense research and to serve as models for resistance, drug-resistant strains of Leishmania tarentolae have been generated in vitro by step-wise exposure to increasing concentrations of antimonials or arsenicals (Borst and Ouellette, Reference Borst and Ouellette1995). A diminished biological reduction of SbV to SbIII has been demonstrated in L. donovani amastigotes resistant to antimony (Shaked-Mishan et al. Reference Shaked-Mishan, Ulrich, Ephros and Zilberstein2001). Aquaglyceroporin1 (AQP1) has recently been demonstrated to mediate uptake of SbIII in Leishmania spp. and their overexpression generates hypersensitivity to SbIII (Marquis et al. Reference Marquis, Gourbal, Rosen, Mukhopadhyay and Ouellette2005). Resistance to antimonials also required an increased synthesis of trypanothione (a bis-glutathionyl-spermidine conjugate), the major intracellular thiol of these parasites (Fairlamb and Cerami, Reference Fairlamb and Cerami1992; Haimeur et al. Reference Haimeur, Brochu, Genest, Papadopoulou and Ouellette2000). Further corroborative evidence was provided by amplification of the GSH1 gene coding for γ-glutamylcysteine synthetase (γ-GCS, Grondin et al. Reference Grondin, Haimeur, Mukhopadhyay, Rosen and Ouellette1997) along with overexpression of ornithine decarboxylase (ODC, Haimeur et al. Reference Haimeur, Guimond, Pilote, Mukhopadhyay, Rosen, Poulin and Ouellette1999), rate-limiting steps in glutathione and spermidine synthesis respectively, and necessary for trypanothione overproduction (Fairlamb and Cerami, Reference Fairlamb and Cerami1992). It was proposed that following the formation of SbIII-thiol complexes (spontaneously or enzymatically), an enhanced extrusion of trivalent heavy metal-thiol conjugates occurs at a rate sufficient to outmatch the influx (Dey et al. Reference Dey, Papadopoulou, Haimeur, Roy, Grondin, Dou, Rosen and Ouellette1994), attributed to an increased expression of P-glycoprotein A or PgpA (Ouellette and Borst, Reference Ouellette and Borst1991), a member of the family of ATP-binding cassette (ABC) proteins, several of which are implicated in drug resistance (Gottesman, Reference Gottesman2002; Lee, Reference Lee2004).
In view of the alarming increase in antimonial resistance in the Indian subcontinent, the next important step would be to elucidate whether these mechanisms are operative in field strains. Singh et al. (Reference Singh, Almeida, Kothari, Kumar, Mandal, Chatterjee, Venkatachalam, Govind, Mandal and Sundar2007), using DNA microarray, have reported amplification of multidrug resistant associated protein A (MRPA) and γ-GCS at a transcriptional level in antimony-resistant clinical isolates. Mukherjee et al. (Reference Mukherjee, Prasad, Singh, Roy, Girard, Chatterjee, Ouellette and Madhubala2007) have reported amplification of MRPA, γ-GCS, and ODC both at a genetic and transcriptional level in one or more antimony-resistant clinical isolates and established that in field isolates, antimony resistance is a multifactorial phenomenon. Accordingly, this study was undertaken in field isolates from the same geographical region, wherein we have focused on the functional aspects of overexpression of γ-GCS and ODC, rate-limiting enzymes of GSH and polyamine biosynthesis respectively. We have been able to establish that in antimony-resistant strains, a higher content of cellular thiols was accompanied by both a decreased generation of reactive oxygen species (ROS) and a higher scavenging activity of ROS, that collectively contribute to decreased antimonial responsiveness.
MATERIALS AND METHODS
Hydrogen peroxide (H2O2, 5%) was obtained from Merck (India). Pentavalent (SbV) and trivalent (SbIII) antimony were kindly provided by Professor S. L. Croft, London School of Tropical Medicine and Hygiene, UK. All other chemicals, unless otherwise mentioned, were obtained from Sigma Chemicals Co. (St Louis, MO, USA).
Parasite culture
Promastigotes of Indian Leishmania donovani strains MHOM/IN/83/AG83, MHOM/IN/90/GE1F8R isolated in 1983 and 1990, respectively, along with 3 untyped strains 2001, NS2 and 41, isolated from patients with visceral leishmaniasis; the strains 2001 and NS2 were isolated in 2000 and 41 were isolated in 2001. These 3 strains showed strong binding with D2, a L. donovani species-specific monoclonal antibody as determined by ELISA (Jaffe and McMahon Pratt, Reference Jaffe and McMahon-Pratt1987; Chatterjee M., personal communication). Promastigotes were routinely cultured at 24°C in M199 medium supplemented with 10% heat-inactivated fetal calf serum (HIFCS) and penicillin/streptomycin referred to as Medium A. For experimental purposes, log phase promastigotes were obtained by subculturing every 72–96 h, the inoculum being 1×106/ml.
Previous studies in the amastigote-macrophage model have established that MHOM/IN/83/AG83 and 2001 are antimony sensitive or Sb-S referred to in this study as S1 and S2 respectively and their IC50 as previously determined was 9·0±0·5 μg/ml and 13·0±1·5 μg/ml; NS2, 41 and MHOM/IN/90/GE1F8R, are antimony resistant or Sb-R referred to as R1, R2 and R3 respectively had IC50 of 24±1·4 μg/ml (R1), 65±3·4 μg/ml (R2) and >100 μg/ml (R3, Mukherjee et al. Reference Mukherjee, Prasad, Singh, Roy, Girard, Chatterjee, Ouellette and Madhubala2007). SAG contains pentavalent antimony (SbV) with m-chlorocresol as preservative which itself is a potent anti-promastigote agent (Roberts and Rainey, Reference Roberts and Rainey1993). Accordingly, in this study, we have used m-chlorocresol-free SbV and SbIII.
Analysis of intracellular thiols using HPLC
Mid log phase promastigotes (5×107) were collected by centrifugation (1600 g, 10 min, 4°C) and derivatized with monobromobimane as described previously (Shim and Fairlamb, Reference Shim and Fairlamb1988). Acid-soluble thiols were separated by ion-paired, reverse-phase HPLC on a Beckman Ultrasphere C18 column using a Beckman System Gold instrument fitted with a Gilson-121 fluorimeter.
Flow cytometric determination of intracellular non-protein thiols using mercury orange (MO)
Non-protein thiols were measured using the method described by O'Connor et al. (Reference O'Connor, Kimler, Morgan and Tempas1988), with slight modifications. The assay was initially standardized for log phase promastigotes (1×107/ml) of S1 and R3, representative of Sb-S and Sb-R strains respectively. After washing cells with ice-cold phosphate buffered saline (0·02 m pH 7·2, PBS), cell pellets were resuspended in mercury orange (100, 250 and 500 μm in acetone) and incubated for 5 min on ice. The cells were then washed thoroughly with ice-cold PBS and analysed for fluorescence in the FL3 channel. Based on the above experiment, we chose 500 μm and log phase promastigotes (1×107/ml) from 5 strains were labelled with mercury orange (500 μm) and MFC analysed as described above.
To measure the regeneration rate of intracellular thiols in promastigotes, parasites (1×106/ml) were initially exposed to buthionine sulphoximine (3 mm, BSO), an inhibitor of γ-GCS (Griffith and Meister, Reference Griffith and Meister1979) for 48 h at 24°C in Medium A. Cells were then washed with PBS, resuspended in Medium A, and the fluorescence of mercury orange was measured to confirm depletion of thiols. Subsequently, cells were harvested at 0, 30, 60 and 150 min and thiol content was assessed. Results were expressed as the percentage increment of MFC following their incubation in BSO-free medium. Similarly, the effect of SbIII (300 μg/ml, 3 h at 37°C) on intracellular thiols was measured in log phase promastigotes (1×106/ml) using mercury orange and results expressed as percentage change of MFC from 0 min.
Effects of pentavalent (SbV) or trivalent (SbIII) antimony on generation of ROS by promastigotes
To study the capacity of SbV and SbIII to generate ROS, log phase promastigotes (1×106/ml) were exposed to SbV or SbIII (300 μg/ml) for 3 h at 37°C in medium M199 (serum free). The cells were then washed with PBS, incubated with dichlorodihydrofluorescein diacetate (H2DCFDA, 50 μm) for 45 min at 37°C and analysed on a FACS Calibur (Becton Dickenson, USA), dead cells being excluded using propidium iodide (PI, 1 μg/ml).
To study whether thiol depletion influenced ROS generated by SbIII, log phase promastigotes from 5 strains were initially treated with BSO (3 mm) to deplete thiols and was confirmed by reduction of fluorescence using mercury orange. These thiol-depleted cells (1×106/ml) were then exposed to SbIII (300 μg/ml) and the extent of ROS generation was measured using H2DCFDA (50 μm), dead cells being excluded using PI.
ROS scavenging activity of promastigotes
To assess the ROS scavenging activity of promastigotes, log phase parasites (1×106/ml) were exposed to H2O2 (1–1000 μm) at 37°C in PBS for 1 h. Cells were then washed and incubated at 37°C for 45 min with H2DCFDA (50 μm). Labelled cells were analysed on a FACS Calibur, dead cells being excluded using PI.
Flow cytometry
Cells (106) from different experimental groups were monitored for their intracellular fluorescence on a flow cytometer (FACS Calibur, Becton Dickenson, San Jose, CA, USA) equipped with an argon-ion laser (15 mW) tuned to 488 nm. Fluorescence of DCF was collected in FL1 channel, equipped with a 530/30 nm band pass filter, PI in FL2 channel having a 585/42-nm band pass filter and mercury orange in FL3 channel equipped with 670 nm long pass filter. Fluorescence was measured in the log mode and expressed as mean fluorescence channel (MFC). Analyses were performed on 10 000 gated events, while data acquisition and analysis was carried out with CellQuest Pro software.
Statistical analysis
Each experiment was performed at least thrice in duplicates and results expressed as mean±standard error of the mean (s.e.m.). For significance, Student's t-test was performed, P values <0·05 were considered significant.
RESULTS
Sb-R strains have higher amounts of cellular thiols than Sb-S strains
Non-protein thiols are established molecules that combat against xenobiotic toxicity and oxidative damage (Meister and Anderson, Reference Meister and Anderson1983). In Leishmaniasis, Mukhopadhyay et al. (Reference Mukhopadhyay, Dey, Xu, Gage, Lightbody, Ouellette and Rosen1996) have shown that in laboratory raised antimonal-resistant L. tarentolae, the degree of drug resistance correlated with concomitantly raised thiol levels. Accordingly, we studied whether differences existed between levels of thiols in Sb-S vs Sb-R field isolates, as measured by HPLC and flow cytometry.
HPLC measurement of thiols, glutathione (GSH) and trypanothione (TSH) indicated that Sb-S strains (S1 and S2) have significantly lower amounts of GSH and TSH than Sb-R strains, R1, R2 and R3 (Table 1) wherein the difference in GSH levels was more pronounced.
Table 1. Thiol levels in Sb-S and Sb-R strains
(Mid log phase promastigotes (5×107) from Sb-sensitive strains (S1 and S2) and Sb-resistant strains (R1, R2 and R3) were derivatized with monobromobimane (Shim and Fairlamb, Reference Shim and Fairlamb1988) and analysed as described in the Materials and Methods section. Each data-point is the mean of at least 3 determinations±s.d.)

* P=<0·001 when compared to the sensitive group.
Mercury orange reacts with all sulfhydryl (-SH) groups, generating a fluorescent product that is retained within cells. However, as the reaction rate of mercury orange with non-protein thiols is much faster than protein thiols, pulse labelling for 5 min on ice allowed MO to react only with non-protein –SH groups. Therefore, the level of fluorescence represented the level of cellular non-protein thiols (O'Connor et al. Reference O'Connor, Kimler, Morgan and Tempas1988). The assay was initially optimized for Leishmania promastigotes using increasing concentrations of MO (100–500 μm). At 100 μm of MO, fluorescence from both the Sb-S (S1) and Sb-R strain (R3) was comparable, MFC being 39·66 and 50·84 respectively. Increasing MO to 250 μm, the MFC of R3 became 2-fold higher than S1 being 92·59 vs 43·95 respectively. A further increase of MO to 500 μm resulted in the MFC of R3 becoming 3-fold higher than S1 being 139·91 vs 45·15 respectively (Fig. 1A). However, further increase in MO caused no change in both strains (data not shown) and accordingly, 500 μm of MO was subsequently selected.

Fig. 1. (A) Flow cytometric measurement of thiols using Mercury Orange (MO). Log phase promastigotes (unstained, a) of S1 (b) and R3 (c) were exposed to MO (500 μm) in acetone, incubated on ice for 5 min, washed and analysed for fluorescence in FL3 channel as described in the Materials and Methods section. (B) Flow cytometric detection of basal intracellular thiols in Sb-S and Sb-R strains. Log phase promastigotes both from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains were labelled with Mercury Orange (MO) before (open bars, □) and after treatment with buthionine sulphoximine (3 mm, 48 h, 24°C) (filled bars, ■) and fluorescence analysed as described in the Materials and Methods section. Data are expressed as MFC±s.e.m. for at least 3 independent experiments in duplicate.
Measurement of non-protein thiols in the 5 strains, as shown in Fig. 1B, clearly indicated that Sb-S strains had lower amounts of thiols, the MFC±s.e.m. of S1 and S2 being 45·24±4·19 and 48·81±3·8 respectively as compared to the Sb-R strains namely R1 (62·09±2·60), R2 (92·33±3·7) and R3 (110·84±7·63). On an individual basis, thiol levels in S1 were lower than the 3 Sb-R strains, P<0·005; a similar trend was observed with S2 in that the thiol levels were significantly lower than R1 (*P<0·01), R2 and R3 (P<0·001). The addition of BSO (3 mm, 48 h) caused a dramatic decrease in fluorescence, confirming that depletion of thiols and the extent of decrease was similar in all strains (Fig. 1B).
Generation of ROS by SbIII is reduced in Sb-R strains
Mehta and Shaha (Reference Mehta and Shaha2006) have shown that antimony exerts its anti-leishmanial activity by generating ROS, that is triggered by loss of mitochondrial membrane potential and uncoupling of oxidative phosphorylation. To examine whether differences exist in the amount of ROS generated by Sb-S vs Sb-R strains in the presence of SbIII, their oxidative status was measured using H2DCFDA. H2DCFDA, a lipid-soluble, membrane-permeable compound is cleaved by non-specific esterases to remove the diacetate portion and release H2DCF, which in turn is oxidized by intracellular reactive oxygen species (ROS) to produce a fluorescent compound DCF (Wan et al. Reference Wan, Myung and Lau1993). Therefore, the fluorescence is directly proportional to the amount of ROS present within cells and H2DCFDA (50 μm) incubated at 37°C for 45 min gave the best results (Fig. 2A).

Fig. 2. (A) H2O2 mediated increase of H2DCFDA mediated fluorescence. Log phase promastigotes of S1 (unstained, a) were exposed to increasing concentrations of H2O2, 100 μm (b) and 1000 μm (c), probed with dichlorodihydrofluorescein diacetate (H2DCFDA) and analysed for dichlorofluorescein (DCF) fluorescence in FL1 channel as described in the Materials and Methods section. (B) Generation of ROS by SbIII and SbV in Sb-S and Sb-R strains. Log phase promastigotes (open bars, □) both from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains were exposed to either SbIII (filled bars, ■) or SbV (hatched bars,
) for 3 h at 37°C, labelled with dichlorodihydrofluorescein diacetate (H2DCFDA) and dichlorofluorescein (DCF) fluorescence was analysed as described in the Materials and Methods section. Data are expressed as MFC±s.e.m. of at least 3 independent experiments in duplicate.
Initially, the concentration of SbIII and duration of incubation was defined by exposing a Sb-S (S1) and a Sb-R strain (R3) to both, variable concentrations of SbIII (0–600 μg/ml) and time (1–6 h) at 37°C. It was established that maximal ROS generation was achieved with SbIII (300 μg/ml) for 3 h, parasite viability remaining >80% as measured by PI uptake (data not shown).
In the Sb-S strains, generation of ROS by SbIII was higher, maximum being in S1 (167·45±20·65) followed by S2 (87·89±12·48) whereas in all 3 Sb-R strains, the amount of ROS generated as compared to S1 was significantly lower, the MFC±s.e.m. being 25·85±5·75, 26·79±6·99 and 64·37±10·54 in R1, R2 and R3 respectively, P<0·001 (Fig. 2B). With regard to S2, the MFC±s.e.m. was significantly higher than R1 and R2 (P<0·001) but was comparable with R3 (P=0·17). Importantly, promastigotes exposed to SbV (300 μg/ml) for 3 h at 37°C failed to generate any ROS, corroborating the earlier reports that SbV is ineffective against promastigotes. Based on this finding we decided to use only SbIII for further experiments.
The rate of thiol synthesis in Sb-S strains is slower than in Sb-R strains
As non-protein thiols were higher in Sb-R strains (Fig. 1B) and were associated with a lower generation of ROS by SbIII in these strains (Fig. 2B), it prompted us to study whether Sb-R strains had an up-regulation in the biosynthetic machinery of thiols which caused a sustained increase in intracellular thiols. Accordingly, the regeneration rate of thiols in Sb-S and Sb-R strains was studied following its depletion using BSO, an established inhibitor of γ-GCS. It was clearly evident that R3 possessed the most efficient thiol-generating machinery, as normal thiol levels were again achieved within 30 min, post-BSO treatment (Fig. 3). The other 2 Sb-R strains, R1 and R2 took 60 min to reach their normal levels and in fact with R2, levels overshot beyond basal levels. In sharp contrast, in the Sb-S strains S1 and S2, their thiol-regenerating machinery was much slower than their Sb-R counterparts, as normal thiol levels were achieved much later at 150 min, post-BSO treatment (Fig. 3).

Fig. 3. Kinetics of thiol regeneration in Sb-S and Sb-R strains. Log phase promastigotes from Sb-S strains, S1 (closed squares, -■-) and S2 (open squares, -□-), as also Sb-R strains R1 (inverted closed triangles, -▼-), R2 (open circles, -○-) and R3 (upright closed triangles, -▲-) were depleted of thiols using buthionine sulphoximine (3 mm, 48 h, 24°C); promastigotes were then washed and resuspended in Medium A, harvested at different time-points, labelled with Mercury Orange (MO) and fluorescence analysed as described in the Materials and Methods section. Data are expressed as mean±s.e.m. of the percentage increment of MFC from 0 min of at least 3 independent experiments in duplicate.
Depletion of intracellular thiols enhances SbIII mediated generation of ROS in Sb-R strains
To confirm that the higher amount of thiols present in Sb-R strains contributes to the attenuated generation of ROS by SbIII, the amount of ROS generated by SbIII following removal of thiols by BSO was measured. In the absence of BSO, SbIII triggered a higher generation of ROS in S1 and S2 than all 3 Sb-R strains, corroborating with previous data. Following thiol depletion by BSO as confirmed by MO (Fig. 1B), ROS production by SbIII was marginally increased in S1, S2 and R3 (Fig. 4). However, in R1 and R2, depletion of thiols resulted in a dramatic 3-fold increase in ROS generation as compared to their respective control values, P<0·001 (Fig. 4). Taken together, removal of thiols failed to augment SbIII-mediated ROS generation in Sb-S strains whereas in Sb-R strains, depletion of thiols triggered a significant increase in fluorescence in R1 and R2 (P<0·001) but not in R3 (P=0·19).

Fig. 4. Effect of thiol depletion on SbIII mediated ROS generation in Sb-S and Sb-R strains. Log phase promastigotes (open bars, □) from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains following removal of thiols (filled bars, ■) were exposed to SbIII (300 μg/ml) for 3 h at 37°C. Cells were labelled with dichlorodihydrofluorescein diacetate (H2DCFDA) and dichlorofluorescein (DCF) fluorescence analysed as described in the Materials and Methods section. Data were expressed as MFC±s.e.m. of at least 3 independent experiments in duplicate.
SbIII depletes thiols primarily in Sb-S strains
Wyllie et al. (Reference Wyllie, Cunningham and Fairlamb2004) have demonstrated that antimony exerts its leishmanicidal activity by depleting thiols causing an altered redox potential. To study whether the amount of thiols depleted by SbIII varied in Sb-S vs Sb-R strains, intracellular thiols were measured before and after treatment with SbIII. In both Sb-S strains, a 3 h incubation with SbIII resulted in an almost 50% depletion of thiols; conversely in all Sb-R strains, SbIII caused minimal change in their thiol levels (Fig. 5).

Fig. 5. Effect of SbIII on thiol levels in Sb-S and Sb-R strains. Log phase promastigotes from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) were exposed to SbIII (300 μg/ml) at 37°C for 3 h; they were then harvested, labelled with Mercury Orange (MO) and fluorescence analysed as described in the Materials and Methods section. Data were expressed as mean±s.e.m. of percentage retention of MFC from baseline of at least 3 independent experiments in duplicate.
Sb-R strains scavenge ROS more efficiently than Sb-S strains
ROS, generated within phagolysosomes contributes significantly to the microbicidal activity of macrophages, H2O2 being one of the major components. With reference to leishmaniasis, Mookerjee Basu et al. (Reference Mookerjee Basu, Mookerjee, Sen, Bhaumik, Sen, Banerjee, Naskar, Choudhuri, Saha, Raha and Roy2006) have established that the leishmanicidal activity of antimony is augmented via enhanced generation of ROS within phagolysosomes. Therefore, it is conceivable that in the antimony resistance phenotype, the Leishmania parasite enhances scavenging of ROS within parasites and/or macrophages.
Measurement of ROS scavenged in Sb-S and Sb-R strains revealed that lower concentrations of H2O2 (1–100 μm) showed no detectable fluorescence, suggesting that Leishmania parasites irrespective of their chemosensitivity profiles, can effectively scavenge this amount of H2O2. However, with increasing concentrations of H2O2, a dose-dependent increase in fluorescence was demonstrated exclusively in Sb-S strains, the average MFC±s.e.m. of S1 and S2 in the presence of 1 mm H2O2 being 1571·63±77·18 and 1462·68±144·36 respectively (Fig. 6). However, in Sb-R strains, addition of 1 mm H2O2 generated a significantly lower fluorescence, the MFC±s.e.m. of R1 and R3 being 272·48±34·78 and 269·21±25·46 respectively (P<0·001) as compared to S1 and S2, indicating that Sb-R strains possess a greater ability to scavenge ROS. With regard to R2, the scavenging of ROS was even more remarkable as with 1 mM of H2O2, the MFC±s.e.m. was only 79·08±4·24, P<0·001 (Fig. 6).

Fig. 6. Flow cytometric detection of ROS-scavenging activity in Sb-S and Sb-R strains. Log phase promastigotes from Sb-S strains S1 (closed squares, -■-), S2 (open squares, -□-) and Sb-R strains R1 (inverted closed triangles, -▼-), R2 (upright open triangles, -△-) and R3 (open circles, -○-) were exposed to increasing concentration of H2O2 (0–1000 μm) at 37°C for 1 h, probed with dichlorodihydrofluorescein diacetate (H2DCFDA) at 37°C for 45 min and dichlorofluorescein (DCF) fluorescence was analysed as described in the Materials and Methods section. Data were expressed as MFC±s.e.m. of at least 3 independent experiments in duplicate.
DISCUSSION
The growing resistance against conventional antimonial drugs is a major problem in the current scenario of Indian visceral leishmaniasis. The primary aim of this study was to investigate the role of anti-oxidative mechanisms contributing to development of antimonial resistance in field isolates. The key findings are that promastigotes from Sb-R field isolates (i) have a higher content of non-protein thiols (ii) curtail SbIII-mediated ROS production and (iii) are more efficient scavengers of ROS than their Sb-S counterparts.
Although antimonial compounds have been used as anti-leishmanial agents for more than 60 years, their mechanism(s) of action is still to be precisely defined. It is an established fact that pentavalent antimony (SbV) is the pro-drug that is reduced to the trivalent (SbIII) form to be an effective anti-leishmanial agent (Sereno et al. Reference Sereno, Cavaleyra, Zemzoumi, Maquaire, Ouaissi and Lemesre1998). This is possibly because promastigotes lack antimony reductase (Shaked-Mishan et al. Reference Shaked-Mishan, Ulrich, Ephros and Zilberstein2001) leading to ineffectiveness of SbV. Accordingly, to simulate in vivo conditions, we have used SbIII in this study. It has been proposed that antimony acts upon several targets that include influencing the bioenergetics of Leishmania parasites by inhibiting parasite glycolysis, fatty acid beta-oxidation and inhibition of ADP phosphorylation (Sundar and Chatterjee, Reference Sundar and Chatterjee2006). It causes non-specific blocking of SH groups of amastigote proteins and inhibition of DNA Topoisomerase I (Chakraborty and Majumder, Reference Chakraborty and Majumder1988). More recently, it has been demonstrated that antimony can compromise the thiol-redox potential in both forms of the parasite by actively promoting efflux of thiols, glutathione and trypanothione and additionally by increasing the proportion of thiols present in their respective disulfide forms. Collectively, antimony acts as a double-edged sword by reducing both the intracellular thiol buffering capacity and the thiol redox potential rendering the parasite more susceptible to oxidative stress (Wyllie et al. Reference Wyllie, Cunningham and Fairlamb2004).
Non-protein thiols, such as glutathione, are well-established molecules for combating oxidative damage, toxic effects of xenobiotics and also for maintaining cellular redox homeostasis (Meister and Anderson, Reference Meister and Anderson1983). Accordingly, augmentation in cellular thiols can attenuate oxidative damage and minimize toxicity of xenobiotics. Trypanosomatids that include Leishmania are unique in that they possess trypanothione [N 1,N 8-bis-(glutathionyl) spermidine], as the major substitute of glutathione present in other systems (Fairlamb et al. 1985). It has been shown that trypanothione is the key intermediate in the regulation of parasite redox homeostasis as well as in defence against xenobiotics and oxidative stress (Fairlamb and Cerami, Reference Fairlamb and Cerami1992; Wyllie et al. Reference Wyllie, Cunningham and Fairlamb2004). Sb-R strains had a significantly higher level of cellular thiols than Sb-S strains, as measured by HPLC and flow cytometry. Additionally, these Sb-R strains have a more efficient biosynthetic machinery for regeneration of thiols than their Sb-S counterparts, as following depletion of thiols, they reverted to their normal level of thiols rapidly within 30–60 min as compared to 150 min required by Sb-S strains. Our previous study also demonstrated that although there is no amplification of γ-GCS at the genetic level in strains (S1, S2, R1, R2 and R3), R2 showed up-regulation at transcriptional level. With regard to ODC, all resistant strains (R1, R2 and R3) showed up-regulation both at genomic and protein level (Mukherjee et al. Reference Mukherjee, Prasad, Singh, Roy, Girard, Chatterjee, Ouellette and Madhubala2007). Taken together, Sb-R strains have a greater potential to curtail oxidative damage and xenobiotic toxicity by sustaining their intracellular thiols.
Mehta and Shaha (Reference Mehta and Shaha2006) have demonstrated that trivalent antimony in promastigotes induces mitochondrial dysfunction and leads to uncoupling of oxidative phosphorylation, ultimately resulting in generation of reactive oxygen species (ROS), the critical effector molecule responsible for parasiticidal activity. In Sb-S strains, it was observed that SbIII triggered a significantly higher amount of ROS production concomitant with a greater proportion of thiol loss than Sb-R strains. This generated a higher level of oxidative stress, accounting for the more potent leishmanicidal activity observed in Sb-S strains. It is known that trivalent antimony is the active form of the drug and absence of DCF fluorescence upon addition of pentavalent antimony to promastigotes corroborated this existing knowledge that pentavalent antimony is ineffective against promastigotes. Additionally, it highlighted that generation of ROS is critical for antimony to mediate its anti-leishmanial activity. It is very likely that this attenuation of SbIII-mediated ROS production in Sb-R strains was achieved by their higher proportion of intracellular thiols. The vital contribution of thiols in the generation of antimony resistance was further substantiated by the selective augmentation of ROS production in Sb-resistant strains, R1 and R2 following removal of thiols. This augmentation in SbIII mediated ROS production following thiol depletion in R1 and R2 negated the possibility of reduced SbIII uptake by AQP1 down-regulation contributing to antimonial resistance. However, in R3, the removal of thiols marginally enhanced generation of ROS suggesting that down-regulation of AQP1 could well be a contributory factor (Marquis et al. Reference Marquis, Gourbal, Rosen, Mukhopadhyay and Ouellette2005). Alternatively, in R3, it is also possible that the rate of regeneration of thiols is rapid enough to efficiently replenish its thiols. The enzyme γ-glutamyl cysteine synthetase (γ-GCS) catalyses conjugation of L-glutamate with L-cysteine and is the rate-limiting step for glutathione biosynthesis (Lu, Reference Lu2000). The activity of γ-GCS is controlled by a non-allosteric feedback by glutathione, cysteine availability, and by factors that control the transcription and post-translational modification of the enzyme (Lu, Reference Lu2000). Considering that in R3, the rate of thiol regeneration was the fastest and it possessed the highest amount of GSH and T[SH]2 and, as Mukherjee et al. (Reference Mukherjee, Prasad, Singh, Roy, Girard, Chatterjee, Ouellette and Madhubala2007) have demonstrated that in these strains, there is only ODC up-regulation with no accompanying γ-GCS upregulation, one is tempted to suggest that in Sb-R strains such as R3, modifications in their γ-GCS activity could well be the key factor for these parasites to become resistant; such studies are ongoing.
Intracellular parasitic protozoans of the genus Leishmania evade toxic, free-radical damage inflicted by the phagocytic macrophage via elaboration of enzymatic pathways such as superoxide dismutase (Ghosh et al. Reference Ghosh, Goswami and Adhya2003), non-enzymatic pathways like non-protein thiols and, importantly, preventing stimulation of macrophages. Antimonial compounds generated ROS (mainly H2O2) within phagolysosomes of macrophages via phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) Ras and extracellular-signal regulated kinase (ERK) (Mookerjee Basu et al. Reference Mookerjee Basu, Mookerjee, Sen, Bhaumik, Sen, Banerjee, Naskar, Choudhuri, Saha, Raha and Roy2006). As inhibition of these proteins or addition of a free-radical scavenger like N-acetylcysteine inhibited antimony-mediated killing of intracellular amastigotes, it emphasized the contribution of ROS in antimony-mediated parasiticidal activity. Therefore, it is evident that the efficacy of antimony as an anti-leishmanial agent hinges upon its ability to generate ROS both within the parasite and/or phagolysosomes of infected macrophages. It would be rational to suggest that Leishmania strains having a higher amount of thiols would also possess a higher capacity to scavenge free radicals and are more likely to be unresponsive to antimony. Here we demonstrate for the first time, that indeed in promastigotes from Sb-S field strains, the addition of H2O2 (1 mm) triggered an almost 7-fold increase in DCF fluorescence as compared to Sb-R strains clearly indicating that the Sb-R strains have a more potent ROS-scavenging activity. This is the first demonstration that in field isolates from patients with VL, Leishmania parasites upregulate their antioxidant pathways through raised non-protein thiols leading to a higher ROS-scavenging activity thus generating antimonial unresponsiveness.
This work received financial assistance from the Council of Scientific and Industrial Research, Government of India and the Wellcome Trust, UK.