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• W2059774444 abstract "The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (SbV). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate SbV and SbIIIvia separate transport systems. Stage-specific intracellular SbV reducing activity was apparent in amastigotes, which reduced the negligibly toxic SbV to highly toxic SbIII. This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to SbV correlates with its level of SbV reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, SbV inhibited the toxicity of SbIII but not of AsIII. Both SbVand SbIII were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes SbVinhibits SbIII but not AsIII activity. Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that SbV anti-leishmanial activity is dependent on its reduction to SbIII. The mechanism of this novel intracellular SbV reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular SbV reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell. The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (SbV). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate SbV and SbIIIvia separate transport systems. Stage-specific intracellular SbV reducing activity was apparent in amastigotes, which reduced the negligibly toxic SbV to highly toxic SbIII. This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to SbV correlates with its level of SbV reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, SbV inhibited the toxicity of SbIII but not of AsIII. Both SbVand SbIII were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes SbVinhibits SbIII but not AsIII activity. Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that SbV anti-leishmanial activity is dependent on its reduction to SbIII. The mechanism of this novel intracellular SbV reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular SbV reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell. flow injection-inductively coupled plasma mass-spectrometry flow injection-hydride generation-inductively coupled plasma-mass spectrometry ion-chromatography-inductively coupled plasma-mass-spectrometry wild type high performance liquid chromatography lethal dose Leishmania donovani is the major causative agent of visceral leishmaniasis. In nature, the parasite exists either as an extracellular promastigote found in the alimentary tract of sandflies or as an obligatory intracellular amastigote found in phagolysosomes of mammalian macrophages(1–3). During the last several years, a number of laboratories have utilized axenic culture of L. donovani amastigotes for the direct evaluation of cell biological and biochemical processes in the amastigote, devoid of the host macrophage (4–7,8,9,10). This technique has also been used to investigate the mechanism of drug action and resistance as well as for screening of potential new drugs(11–13). The treatment of choice of human visceral leishmaniasis is the administration of pentavalent antimony (SbV)-containing drugs such as sodium stibogluconate (Pentostam, Wellcome, Beckenham, UK) or meglumine antimoniate (Glucantime, Rhone-Poulenc, Paris, France). Despite extensive use of these compounds over the last decades, the mechanism of action remains unclear. It has been hypothesized that SbV is not toxic toLeishmania, but rather that it is enzymatically reduced, probably by the host macrophage, to SbIII, the form of antimony that is highly Leishmania-toxic (14Goodwin L.G. Trans. R. Soc. Trop. Med. Hyg. 1995; 89: 339-341Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 15Goodwin L.G. Page J.E. Biochem. J. 1943; 22: 236-240Google Scholar, 16Berman J.D. Waddell D. Hanson B.D. Antimicrob. Agents Chemother. 1985; 27: 916-920Crossref PubMed Scopus (108) Google Scholar, 17Roberts W.L. Berman J.D. Rainey P.M. Antimicrob. Agents Chemother. 1995; 39: 1234-1239Crossref PubMed Scopus (123) Google Scholar, 18Roberts W.L. Rainey P.M. Antimicrob. Agents Chemother. 1993; 37: 1842-1846Crossref PubMed Scopus (58) Google Scholar). In contrast, it has been shown that SbV is directly toxic to axenic amastigotes (12Ephros M. Bitnun A. Shaked P. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1999; 43: 278-282Crossref PubMed Google Scholar, 13Ephros M. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1997; 41: 1064-1068Crossref PubMed Google Scholar, 19Callahan H.L. Portal A.C. Devereaux R. Grogl M. Antimicrob. Agents Chemother. 1997; 41: 818-822Crossref PubMed Google Scholar), thus negating the necessity of the macrophage for SbV reduction. Furthermore, these data imply that either the parasite reduces SbV to SbIIIintracellularly, and thus SbIII is directly toxic to amastigotes, or that both oxidation states of antimony are active against Leishmania amastigotes. The modes of action of the two oxidation states of antimony (SbIII and SbV) on Leishmania are, as yet, not fully understood. Several groups have shown obligatory cross-resistance between SbV, SbIII, and arsenite (AsIII) inLeishmania tarentolae, Leishmania major,Leishmania mexicana, L. donovani and Leishmania infantum (11Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Crossref PubMed Google Scholar, 20Haimeur A. Ouellette M. Antimicrob. Agents Chemother. 1998; 42: 1689-1694Crossref PubMed Google Scholar, 21Dey S. Papadopoulou B. Roy G. Grondin K. Dou D. Rosen B.P. Ouellette M. Haimeur A. Mol. Biochem. Parasitol. 1994; 67: 49-57Crossref PubMed Scopus (102) Google Scholar, 22Borst P. Ouellette M. Annu. Rev. Microbiol. 1995; 49: 427-460Crossref PubMed Scopus (267) Google Scholar). In contrast, it has been suggested that, at least in L. donovani, such cross-resistance does not necessarily occur (12Ephros M. Bitnun A. Shaked P. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1999; 43: 278-282Crossref PubMed Google Scholar). Furthermore, it has been demonstrated that, when bound to organic compounds, structural similarities exist between SbIII and SbV (23Roberts W.L. McMurray W.J. Rainey P.M. Antimicrob. Agents Chemother. 1998; 42: 1076-1082Crossref PubMed Google Scholar). For example, the trivalent antimony compound potassium antimonyl tartrate has a structure resembling that of the pentavalent antimony compound sodium stibogluconate. Thus it is conceivable that SbV and SbIII act on a common parasite target molecule. It is also possible that, when given together, these compounds might act additively or synergistically, or might inhibit one another. It is also possible that at least part of the anti-leishmanial activity of AsIII may not be mediated by a mechanism similar to that of antimony despite the fact that cross-resistance in otherLeishmania species has been observed (11,20–22). To understand the relationships between these compounds and their activity against L. donovani, antimony accumulation and its subsequent intracellular metabolism were investigated using novel antimony speciation methods. Sodium stibogluconate (powder) was a gift from The Wellcome Trust (Beckenham, UK). Potassium antimonyl tartrate was obtained from Sigma Chemical Co. (St. Louis, MO.). [3H]Thymidine was obtained from PerkinElmer Life Sciences. Medium 199 and fetal calf serum were obtained from Biological Industries, Inc. (Bet Haemeek, Israel). Materials for antimony determinations were as follows: sodium tetrahydridoborate (puriss. p.a. for determination of hydride formers) and potassium fluoride (MicroSelect; >99.5% purity) were obtained from Fluka, Inc. (Seelze, Germany), potassium iodide (puriss, p.a.; >99% purity) was obtained from Sigma-Aldrich, Inc. (Deisenhofen, Germany). All other chemicals were of analytical grade. A cloned line of L.donovani 1SR (5,24) and the Pentostam-resistant mutant L. donovani Ld1S.20 (13Ephros M. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1997; 41: 1064-1068Crossref PubMed Google Scholar) were used. Promastigotes were grown in medium 199 at 26 °C and supplemented with 10% fetal calf serum. In vitro culture of amastigotes was performed as described by Saar et al. (5Saar Y. Asamoa R. Waldman E. Mazareb S. Amin-Spector S. Plumblee J. Turco S.J. Zilberstein D. Mol. Biochem. Parasitol. 1998; 95: 9-20Crossref PubMed Scopus (147) Google Scholar). Transformation of amastigotes to promastigotes was performed by centrifugation of amastigotes (1,200 × g, at room temperature for 10 min), suspension in promastigote medium, and incubation at 26 °C. Under these conditions, amastigotes differentiate to promastigotes within 48 h (5Saar Y. Asamoa R. Waldman E. Mazareb S. Amin-Spector S. Plumblee J. Turco S.J. Zilberstein D. Mol. Biochem. Parasitol. 1998; 95: 9-20Crossref PubMed Scopus (147) Google Scholar). Parasite viability was measured using a radiolabeled thymidine incorporation assay as follows: mid-log phase cells (∼5 × 105 cells/ml) were added in duplicate to 24-well flat-bottomed microtiter plates (2-ml final volume). Drugs were serially added to the cells (either promastigotes or amastigotes), and cells were then incubated for 48 h at either 26 °C (promastigotes) or 37 °C in 5% CO2 (amastigotes). Subsequently, 10 μl of [3H]thymidine (0.1 mCi/ml) were added to each well and incubated for another 3 h (promastigotes) or 24 h (amastigotes). 1-ml aliquots from each well were centrifuged (1,200 × g at room temperature for 10 min) and added to 5 ml of ice-cold trichloroacetic acid, vortexed, and incubated on ice for about 20 min. Samples were then filtered through a glass microfiber filter (GF/C, Whatman International Ltd., Springfield Mill, UK) and washed once with 10 ml of cold trichloroacetic acid followed by 10 ml of 95% ethanol. Radioactivity was measured using β-scintillation counting. Results were expressed as percentage viability. L. donovani promastigotes and amastigotes were incubated for 3, 6, or 12 h with potassium antimonyl tartrate (34 μg of SbIII/ml, 0.28 mm SbIII), sodium stibogluconate (1 mg of SbV/ml, 8.2 mm SbV), or both. Subsequently, cells were washed twice with ice-cold phosphate-buffered saline and then extracted with concentrated nitric acid. The content of antimony in each sample was measured using flow injection-inductively coupled plasma-mass spectrometry (FI-ICP-MS)1 atm/z 121 using water as solvent stream (flow rate was 1.5 ml/min, n = 5) under clean room conditions (class 1000). Each sample contained 100-μl aliquots of cell extract, which were diluted 1:100 with water (25Ulrich N. Anal. Chim. Acta. 1998; 359: 245-253Crossref Scopus (85) Google Scholar, 26Ulrich N. Fresenius J. Anal. Chem. 1998; 360: 797-800Crossref Scopus (28) Google Scholar). For each measurement, aliquots of 10 μl were injected with five repetitions. Intracellular SbIII and SbV were measured using flow injection-hydride generation-inductively coupled plasma-mass spectrometry (FI-HG-ICP-MS), which has recently been developed (27Ulrich N. Anal. Chim. Acta. 2000; 417: 201-209Crossref Scopus (37) Google Scholar). Briefly, SbIII was reduced to antimony hydride (SbH3) using 0.2% sodium tetrahydridoborate in de-ionized water and was directly quantified using mass spectrometry at m/z 121. To avoid SbV interference with the antimony hydride measurements, the SbV reduction was suppressed by the addition of 100 mg/liter of fluoride (as KF) to the solvent stream. Subsequently, SbV was reduced to antimony hydride in 2 steps; pre-reduction using 1.2% KI followed by reduction with 0.2% sodium tetrahydridoborate, both in de-ionized water as solvent stream. The SbV content in each sample was calculated as the difference between the absorbance determined after the second reduction (SbIII + SbV) and first reduction (SbIII). Nonreducible organic antimony is not detected by this method. For each measurement, aliquots of 100 μl were injected with five repetitions. Chromatographic separation of intracellular antimony was done as described by Ulrich et al. (28Ulrich N. Shaked-Mishan P. Zilberstein D. Fresenius J. Anal. Chem. 2000; 368: 62-66Crossref PubMed Scopus (28) Google Scholar). Briefly, cell extracts were diluted 1:100 with distilled water, and 100-μl aliquots from each preparation were injected into a laboratory-packed PRP-X100 HPLC column. SbV and SbIII were eluted from the column using 15 mm nitric acid and were directly injected to ICP-MS (m/z 121) for antimony absorbance. Each sample was filtered and degassed prior to chromatography. Antimony accumulation in promastigotes and amastigotes of both the wild-type and the mutant L. donovani Ld1S.20 was determined using two different methods. First, total intracellular antimony content was measured with FI-ICP-MS (25Ulrich N. Anal. Chim. Acta. 1998; 359: 245-253Crossref Scopus (85) Google Scholar, 26Ulrich N. Fresenius J. Anal. Chem. 1998; 360: 797-800Crossref Scopus (28) Google Scholar), and the results are summarized under “Total [Sb]i” in Tables 1 and 2. The second method used to determine the content of antimony hydride (SbH3) was FI-HG-ICP-MS (27Ulrich N. Anal. Chim. Acta. 2000; 417: 201-209Crossref Scopus (37) Google Scholar, 28Ulrich N. Shaked-Mishan P. Zilberstein D. Fresenius J. Anal. Chem. 2000; 368: 62-66Crossref PubMed Scopus (28) Google Scholar). In this method, SbV and SbIII in cell extracts were separately reduced to SbH3, which then flowed through the plasma to the mass spectrometer, where its concentration was subsequently determined (Tables 1 and 2, [SbV]i and [SbIII]i). This method enables the determination of the intracellular content of each of the antimony oxidation states.Table IAccumulation of antimony by L. donovani promastigotesParasitesExtracellular antimonyIntracellular antimony ng Sb/1 × 106 cellsTotal [Sb]i[SbIII]i[SbV]iLd1SR (WT)SbIII1-a34 μg of SbIII/ml, corresponding to 0.28 mM SbIII.50 ± 226 ± 23 ± 1Ld1SRSbV1-b1 mg of SbV/ml, corresponding to 8.2 mM SbV.98 ± 41.5 ± 0.374 ± 4Ld1SRSbIII + SbV183 ± 1229 ± 2127 ± 5Ld1SRNone0.6 ± 0.1Ld1S.20 (mutant)SbIII43 ± 224 ± 21.8 ± 0.2Ld1S.20SbV87 ± 41.3 ± 0.267 ± 3Ld1S.20SbIII + SbV143 ± 625 ± 2106 ± 5Ld1S.20None1.0 ± 0.2Promastigotes were incubated with potassium antimonyl tartrate (SbIII), sodium stibogluconate (SbV) or both for 12 h at 26 °C. Cell extractions and intracellular antimony concentration determinations using FI-ICP-MS (total [Sb]icolumn) and FI-HG-ICP-MS ([SbIII]i and [SbV]i columns) were performed as described under “Experimental Procedures.”1-a 34 μg of SbIII/ml, corresponding to 0.28 mM SbIII.1-b 1 mg of SbV/ml, corresponding to 8.2 mM SbV. Open table in a new tab Table IIAccumulation of antimony by L. donovani amastigotesParasitesExtracellular antimonyIntracellular antimony ng Sb/1 × 106 cellsTotal [Sb]i[SbIII]i[SbV]iLd1SR (WT)SbIII (34 μg/ml)48 ± 242 ± 32.0 ± 0.5Ld1SRSbV (0.2 mg/ml)187 ± 855 ± 4.0103 ± 6Ld1SRSbV (1 mg/ml)653 ± 40118 ± 8376 ± 15Ld1SRSbIII2-a34 μg of SbIII/ml, corresponding to 0.28 mM SbIII. + SbV2-b1 mg of SbV/ml, corresponding to 8.2 mM SbV.890 ± 57179 ± 12638 ± 10Ld1SRNone1.5 ± 0.4Ld1S.20 (mutant)SbIII (34 μg/ml)56 ± 345 ± 31.5 ± 0.5Ld1S.20SbV (200 μg/ml)59 ± 32.5 ± 0.849 ± 3Ld1S.20SbV (1 mg/ml)407 ± 182.9 ± 0.8318 ± 16Ld1S.20SbIII2-a34 μg of SbIII/ml, corresponding to 0.28 mM SbIII. + SbV2-b1 mg of SbV/ml, corresponding to 8.2 mM SbV.548 ± 3915 ± 1445 ± 22Ld1S.20None1.7 ± 0.2Amastigotes were incubated with potassium antimonyl tartrate (SbIII), sodium stibogluconate (SbV) or both for 12 h at 37 °C in 5% CO2. Cell extractions and intracellular antimony concentration determinations using FI-ICP-MS (total [Sb]i column) and FI-HG-ICP-MS ([SbIII]i and [SbV]i columns) were performed as described under “Experimental Procedures.”2-a 34 μg of SbIII/ml, corresponding to 0.28 mM SbIII.2-b 1 mg of SbV/ml, corresponding to 8.2 mM SbV. Open table in a new tab Promastigotes were incubated with potassium antimonyl tartrate (SbIII), sodium stibogluconate (SbV) or both for 12 h at 26 °C. Cell extractions and intracellular antimony concentration determinations using FI-ICP-MS (total [Sb]icolumn) and FI-HG-ICP-MS ([SbIII]i and [SbV]i columns) were performed as described under “Experimental Procedures.” Amastigotes were incubated with potassium antimonyl tartrate (SbIII), sodium stibogluconate (SbV) or both for 12 h at 37 °C in 5% CO2. Cell extractions and intracellular antimony concentration determinations using FI-ICP-MS (total [Sb]i column) and FI-HG-ICP-MS ([SbIII]i and [SbV]i columns) were performed as described under “Experimental Procedures.” As shown in Table 1 (Total [Sb]i), in WT promastigotes, SbIII or SbV each accumulated intracellularly after a 12-h incubation. Furthermore, when WT promastigotes were simultaneously incubated with both SbV and SbIII, the total [Sb] measured intracellularly approximated the sum of the intracellular concentrations of the two antimony oxidation states when incubations were with SbV or SbIII alone. The same was true with promastigotes of the mutant L. donovani Ld1S.20. With a similar set of experiments using WT and mutant amastigotes, the data in Table 2 (Total [Sb]i) show that both SbIII and SbV were accumulated intracellularly. SbV accumulation was concentration-dependent, suggesting that its transport may be mediated by a permease. When compared with promastigotes, the transport of SbV into WT amastigotes is 6.7- and 4.9-fold greater, when incubation was with SbV alone or with both oxidation states concomitantly, respectively. SbIII accumulation was similar in promastigotes and amastigotes of both WT and mutant L. donovani Ld1S.20. The change in SbV accumulation in mutant amastigotes resembles that of WT amastigotes although to a lesser extent, with SbV accumulation increasing by only 4.9-fold. To determine the intracellular fate of antimony in both promastigotes and amastigotes, cell extracts of parasites treated with SbV, SbIII, or both, were analyzed for each of the antimony oxidation states using FI-HG-ICP-MS. The SbVconcentrations used were those that in competition experiments (Figs. 2and 3) resulted in 10 and 100% inhibition of SbIIItoxicity by SbV.Figure 3Effect of sodium stibogluconate (SbV) on the toxicity of potassium antimonyl tartrate (SbIII) or arsenite (AsIII) on L. donovani amastigotes. A, additive toxicity of SbV (sodium stibogluconate) and SbIII(potassium antimonyl tartrate) on wild-type L. donovani amastigotes. Cells were pretreated with either 10 μg/ml potassium antimonyl tartrate (▴, 3.4 μg/ml SbIII) or 100 μg/ml potassium antimonyl tartrate (●, 34 μg/ml SbIII). Viability was determined as described under “Experimental Procedures.” B, effect of sodium stibogluconate on the toxicity of potassium antimonyl tartrate and arsenite on amastigotes of the L. donovani mutant Ld1S.20. Amastigotes were pretreated with either 100 μg/ml potassium antimonyl tartrate (●, 35 g/ml SbIII) or 1 μg/ml arsenite (▪). Viability was determined using [3H]thymidine incorporation. The results are expressed as mean ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In WT promastigotes incubated with either SbIII or SbV alone, only the specific oxidation state added to the extracellular medium was found intracellularly (Table 1, [SbIII]i and [SbV]i). Most of the accumulated SbV (76%) was identified as intracellular SbV, but only 52% of the accumulated SbIII was recovered by FI-HG-ICP-MS as SbIII. When the extracellular medium contained SbIII and SbV, both these forms were found intracellularly as well with similar SbIIIconcentrations. SbV accumulated at a 71% higher concentration when parasites were incubated with SbV and SbIII (versus with SbV alone). Similar results were obtained when these experiments were performed using mutant L. donovani Ld1S.20 promastigotes. In contrast to the above findings, when amastigotes of WT parasites were subjected to the same conditions (Table 2), both SbVand SbIII were detected intracellularly when parasites were incubated with only SbV. Thus, reduction of SbVto SbIII was observed in WT amastigotes incubated with SbV only. When incubated for 12 h with 0.2 mg of SbV/ml, WT amastigotes reduced 29% of the accumulated antimony to SbIII; 18% was reduced when incubation was with 1 mg of SbV/ml. This may indicate that the latter concentration is at saturation for enzymatic SbV reduction. In contrast, only minor levels of reduction of SbV to SbIII in mutant amastigotes occurred. Furthermore, the high level of SbIII accumulation by the mutant amastigotes (Table 2) rules out a possible role for active SbIII efflux in Ld1S.20 resistance to pentavalent antimony. The results in Tables 1 and 2 indicate that SbV is stable. No spontaneous reduction of SbV to SbIII was observed during the 12-h assays. Only 1% reduction was observed after a few weeks (not shown). This is in agreement with previous observations that SbV is stable in aqueous solutions even though the redox potential favors SbIII (29Lugo de Yarbuh A. Anez N. Petit de Pena Y. Burguera J.L. Burguera M. Annu. Trop. Med. Parasitol. 1994; 88: 37-41Crossref PubMed Scopus (21) Google Scholar). Hence, it is likely that rapid reduction of SbV requires intracellular catalytic activity of either an enzymatic or nonenzymatic nature. To further delineate the nature of the intracellular reduction of SbV, [SbIII]i was determined after 3-, 6-, and 12-h incubations of both WT and mutant promastigotes and amastigotes with SbV alone. As shown in Fig.1, WT amastigotes showed a time-dependent reduction of SbV whereas mutant amastigotes exhibited less than 0.025 of the reducing activity. Promastigotes, both WT and mutants, did not reduce SbV to any extent. The results in Fig. 1 and Tables 1 and 2 indicate that SbVreducing activity correlates with the previously documented stage-specific susceptibility of WT amastigotes to pentavalent antimony(12). However, in amastigotes, the intracellular concentration of SbV was 5-fold higher than in promastigotes, suggesting that it may also be toxic to amastigotes. To assess the specific role of SbV in the anti L. donovani activity of antimony, the intracellular relationship between SbV and SbIII was studied with competition assays. Dose response analyses were performed by incubating wild-type L. donovani 1SR with variable concentrations of sodium stibogluconate (SbV) and a fixed potassium antimonyl tartrate (SbIII) concentration equivalent to the LD90for WT L. donovani promastigotes (100 μg/ml = 34 μg of SbIII/ml). As shown in Fig.2 A, [SbV] of ≥ 200 μg/ml neutralized the toxicity of SbIII, resulting in no mortality at 1 mg/ml SbV. Also, the minimal SbV concentration required for reversion of SbIII toxicity declined with decreasing SbIIIconcentration (not shown). A similar relationship between SbV and SbIII was also shown with promastigotes from the Pentostam-resistant mutant L. donovani Ld1S.20 (Fig. 2 B). In contrast, SbV failed to reverse arsenite (AsIII) toxicity to L. donovani even at SbV concentrations as high as 5 mg/ml (Fig. 2, A and B). These data emphasize the specificity of the SbV-SbIII interaction in L. donovani promastigotes, both WT and mutant. For wild-type amastigotes, the dose-response curve for SbVin the presence of a fixed low dose of SbIII (3.4 μg of SbIII/ml, equivalent to the LD20) showed additive toxicity of SbV and SbIII (Fig.3 A). In the presence of 34 μg of SbIII/ml (equivalent to the LD90) no inhibition of SbIII toxicity by SbV was observed. This is in agreement with the previously reported stage-specific susceptibility of L. donovani to SbV (12Ephros M. Bitnun A. Shaked P. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1999; 43: 278-282Crossref PubMed Google Scholar). When the same dose-response analysis was performed using amastigotes of the L. donovani mutant Ld1S.20, SbV inhibited SbIII toxicity in a fashion similar to that observed in promastigotes. As shown in Fig. 3 B, [SbV] ≥ 200 μg/ml reduced mortality caused by high dose SbIII (34 μg/ml SbIII), and at about 1 mg of SbV/ml no mortality was observed. When these mutant amastigotes were treated with AsIII instead of SbIII, SbV failed to reverse AsIII toxicity, further emphasizing the specificity of the SbV-SbIII competition. Differences in antimony intracellular accumulation and reducing activity were observed between promastigotes and amastigotes and between WT and mutant parasites. Furthermore, often total [Sb]i was greater than [SbV + SbIII]i (Tables 1 and 2). Therefore, to ascertain whether antimony metabolism other than oxidation or reduction occurs intracellularly, cell extracts from the above experiments were subjected to anion exchange chromatography, and fractions eluted from the column were directly subjected to ICP-MS. When a parasite-free solution containing both sodium stibogluconate and potassium antimonyl tartrate was subjected to HPLC anion exchange chromatography, two distinct peaks (SbV and SbIII), were identified. SbV and SbIII retention times were 100 and 300 s, respectively (not shown). Fig. 4 shows chromatograms of WT and mutant amastigotes and promastigotes incubated with SbV. As shown, when amastigotes were incubated with SbV, the SbIII peak was absent in the mutant but not in WT. This correlates with the lack of SbVreducing activity observed in the mutant amastigotes (Fig. 1). In both WT and mutant amastigotes, peaks other than free SbV and SbIII were detected. Differences in peaks A through F were apparent between WT and mutant. These peaks might represent covalent interactions of either SbV or SbIII with intracellular molecules. No major differences were observed between WT and mutant promastigotes when incubations were performed with SbV (Fig. 4,C and D). Despite the qualitative nature of the chromatographic results, the size of both the promastigote SbIII peaks (WT and mutant) was smaller than in WT amastigotes, further emphasizing the low level of SbVreducing activity in promastigotes (Fig. 1). Bacteria and yeast that live in environments contaminated with arsenate (AsV) reduce the element intracellularly to arsenite (AsIII) as part of the mechanism that was evolved to evade the toxic effects of this heavy metal (30Rosen B.P. Trends Microbiol. 1999; 7: 207-212Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Given thatLeishmania spp. are not necessarily exposed to heavy metals in their natural habitats (sandflies and vertebrates), they may not have developed this type of protective mechanism. In fact, by reducing nontoxic SbV Leishmania, they actually expose themselves to the lethal effects of SbIII. This work aimed to assess the role of SbV reduction in antimony toxicity to and resistance in L. donovani. The data presented herein show the following. 1) Intracellular stage-specific SbV reduction occurs in WT L. donovani 1SR and susceptibility to SbV correlates with the level of SbV reducing activity. 2) The SbV-resistant mutant amastigote of L. donovani Ld1S.20 is deficient in SbV intracellular reducing activity. 3) Antimony metabolism with stage-specific differences occurs in both promastigotes and amastigotes of WT L. donovani 1SR and 4) stage-specific intracellular competition between SbIII and SbV, but not AsIII, occurs in WT L. donovani 1SR. This competition is not related to antimony transport. The results of the experiments described indicate that SbVreverses the toxicity of SbIII to L. donovani promastigotes, in both the WT and the mutant L. donovani Ld1S.20. In addition, SbV and SbIII are toxic in an additive fashion to WT amastigotes. These data support previous findings that described the stage-specific toxicity of SbV, but not of SbIII, to L. donovani (12Ephros M. Bitnun A. Shaked P. Waldman E. Zilberstein D. Antimicrob. Agents Chemother. 1999; 43: 278-282Crossref PubMed Google Scholar). In contrast, in mutant L. donovani Ld1S.20 amastigotes, a response similar to promastigotes was observed (inhibition). In both wild-type and mutant promastigotes and amastigotes, no cross-inhibition by SbV occurs when AsIII is substituted for SbIII. This suggests that, despite similar activity in other biological systems (30Rosen B.P. Trends Microbiol. 1999; 7: 207-212Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 31Rosen B.P. J. Basic Clin. Physiol. Pharmacol. 1995; 6: 251-263Crossref PubMed Scopus (48) Google Scholar), in L. donovani, antimony and arsenic may not act via similar mechanisms. FI-HG-ICP-MS and FI-ICP-MS data show that both antimony oxidation states, either individually or together, enter promastigotes and axenic amastigotes. The specific quantitative results for combined SbIII and SbV incubation rule out the possibility that competition occurs at the plasma membrane transport level. This is true for both promastigotes and amastigotes of both WT and mutant L. donovani Ld1S.20 parasites. Because SbV-SbIII antagonism does not take place at the transport level, the assumption that it transpires intracellularly has been borne out. Stage-specific intracellularL. donovani SbV reducing activity exists in WT amastigotes, which reduces negligibly toxic SbV to highly toxic SbIII. In WT promastigotes, the putative L. donovani stage-specific antimony reducing activity may be expressed at either a low level or may be of very low activity, thus explaining the negligible susceptibility of these promastigotes to SbV. In the mutant L. donovani Ld1S.20 amastigote SbVreducing activity is present either in small amounts or may be nonexistent; thus SbV accumulates intracellularly but is not reduced to SbIII. Consequently mutant amastigotes are less susceptible than WT to SbV. The data presented in Fig.1 indicate that in WT amastigotes, stage-specific SbVreducing activity occurs, whereas in the mutant amastigote, SbV reduction is less than 0.025 that of WT, thus SbV accumulates intracellularly but is not reduced to SbIII. Furthermore, the observation that both WT and mutant promastigotes and amastigotes accumulate SbIII to the same level (Tables 1 and 2) rules out a possible role for antimonite efflux as the reason for low level of SbIII reduction as well as for Ld1S.20 SbV resistance. The SbV reducing activity of L. donovani might resemble that of the arsenate reductase previously described in bacteria and yeast, which catalyzes reduction of AsV to AsIII (30Rosen B.P. Trends Microbiol. 1999; 7: 207-212Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 32Mukhopadhyay R. Rosen B.P. FEMS Microbiol. Lett. 1998; 168: 127-136Crossref PubMed Google Scholar, 33Liu J. Rosen B.P. J. Biol. Chem. 1997; 272: 21084-21089Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is possible that an L. donovani enzyme that catalyzes SbV reduction plays a key role in parasite susceptibility to antimony. This possibility is currently being investigated in our laboratory. In WT amastigotes, despite SbV reduction, [SbV]i remains relatively high and theoretically should have inhibited the intracellular SbIIIpresent (Table 2), as seen in both WT and mutant promastigotes as well as in mutant amastigotes. However, the data presented in Fig.3 A show that this does not occur. This might suggest that SbV is directly toxic to WT amastigotes. If this is the case, it must be assumed that SbV toxicity is coupled with SbV reducing activity. Fig. 5 illustrates a model describing antimoniate reduction and the subsequent neutralization of SbIII by trypanothione. We propose that SbIIIaccumulates in cells by transport across the plasma membrane and/or by the intracellular reduction of SbV to SbIII via either an enzymatic or nonenzymatic mechanism. SbIII then complexes with trypanothione, and this complex is then extruded from the parasite via specific transporters, as has been previously proposed (20Haimeur A. Ouellette M. Antimicrob. Agents Chemother. 1998; 42: 1689-1694Crossref PubMed Google Scholar, 34Dey S. Ouellette M. Lightbody J. Papadopoulou B. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2192-2197Crossref PubMed Scopus (133) Google Scholar, 35Ouellette M. Legare D. Haimeur A. Grondin K. Ray G. Brochu C. Papadopoulou B. Drug Resistance Updates. 1998; 1: 43-48Crossref PubMed Scopus (61) Google Scholar). A mutation in a purported stage-specific SbVreductase would result in resistance of amastigotes to SbVbut not to SbIII. However, any mutation that occurs in enzymes involved in reactions downstream of the site of the proposed SbV stage-specific reducing activity should result in antimony as well as in arsenite-antimony cross-resistance. This could explain why others have reported SbV-SbIII-AsIII cross-resistance in various Leishmania species (11Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Crossref PubMed Google Scholar, 20Haimeur A. Ouellette M. Antimicrob. Agents Chemother. 1998; 42: 1689-1694Crossref PubMed Google Scholar, 22Borst P. Ouellette M. Annu. Rev. Microbiol. 1995; 49: 427-460Crossref PubMed Scopus (267) Google Scholar, 36Grondin K. Haimeur A. Mukhopadhyay R. Rosen B.P. Ouellette M. EMBO J. 1997; 16: 3057-3065Crossref PubMed Scopus (167) Google Scholar). Furthermore, the additional role of macrophage-associated antimony reduction in natural infection, as previously suggested (11Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Crossref PubMed Google Scholar, 16Berman J.D. Waddell D. Hanson B.D. Antimicrob. Agents Chemother. 1985; 27: 916-920Crossref PubMed Scopus (108) Google Scholar, 17Roberts W.L. Berman J.D. Rainey P.M. Antimicrob. Agents Chemother. 1995; 39: 1234-1239Crossref PubMed Scopus (123) Google Scholar), cannot be ruled out. In promastigotes, the observation that SbV inhibits SbIII toxicity is surprising. No similar phenomenon has been reported before. The intracellular SbV antagonism of SbIII toxicity in WT and mutant promastigotes as well as in mutant amastigotes could occur because of a number of possibilities. In solution, gluconic acid dissociates from antimony and is replaced by hydroxyl groups forming hexahydroxyl antimony, whereas tartrate remains conjugated to SbIII. Thus, SbV antagonism of SbIII is more likely to be a result of noncompetitive inhibition rather than of competitive inhibition (because of structural similarity). Alternatively, an inactive oxide hybrid is formed between potassium antimonyl tartrate and one-half of the sodium stibogluconate molecule. Because SbV is rapidly reduced to SbIII in amastigotes, such hybrids will probably not form. These possibilities are currently being investigated. The recently developed antimony anion exchange chromatography method (28Ulrich N. Shaked-Mishan P. Zilberstein D. Fresenius J. Anal. Chem. 2000; 368: 62-66Crossref PubMed Scopus (28) Google Scholar) showed that SbV is not only reduced but also metabolized by L. donovani. Peaks separated from cell extracts after SbV treatment (Fig. 4) suggest the possibility of covalent interactions of either SbV or SbIII with intracellular molecules. The bound antimony may not be reduced by BH4 (27Ulrich N. Anal. Chim. Acta. 2000; 417: 201-209Crossref Scopus (37) Google Scholar), thus explaining the differences between total [Sb]i and the reducible intracellular SbV and SbIII measured in Tables 1 and 2. The amount of antimony accumulated in L. donovani promastigotes and amastigotes are 20 and 300 times higher than that reported for L. panamensis (17Roberts W.L. Berman J.D. Rainey P.M. Antimicrob. Agents Chemother. 1995; 39: 1234-1239Crossref PubMed Scopus (123) Google Scholar). Furthermore, although this New World Leishmania species also exhibits stage-specific susceptibility to SbV, both developmental stages accumulate it to the same extent. Hence, it is likely that in New and Old World species, SbV acts via different modes of action. The novel approaches of FI-HG-ICP-MS, FI-ICP-MS, and anion exchange chromatography facilitated the description of intracellular antimony metabolism in L. donovani promastigotes and amastigotes, both WT and mutant. To the best of our knowledge, this is the first description of intracellular SbV reduction in parasitic protozoa as well as in any intact cell. We thank Prof. Stephen M. Beverley and Dr. Mark L. Cunningham for useful discussions." @default.
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• W2059774444 title "Novel Intracellular SbV Reducing Activity Correlates with Antimony Susceptibility in Leishmania donovani" @default.
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