Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m405635200
ISSN1083-351X
AutoresSusan Wyllie, Mark L. Cunningham, Alan H. Fairlamb,
Tópico(s)Garlic and Onion Studies
ResumoDespite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (SbIII) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, SbIII decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, SbIII inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance. Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (SbIII) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, SbIII decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, SbIII inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance. Leishmania parasites cause a wide spectrum of human and animal infections ranging from life-threatening visceral disease to disfiguring mucosal and cutaneous forms of the disease. These “neglected” diseases are a significant cause of morbidity and mortality in 88 countries in the Americas, Africa, Asia, and Southern Europe; millions of people are at risk, and 400,000 new cases are reported annually (1Guerin P.J. Olliaro P. Sundar S. Boelaert M. Croft S.L. Desjeux P. Wasunna M.K. Bryceson A.D. Lancet Infect. Dis. 2002; 2: 494-501Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Leishmania spp. are obligate intracellular parasites of the vertebrate reticuloendothelial system, where they multiply as amastigotes in macrophage phagolysosomes; transmission is by blood-sucking sandflies, in which they proliferate as extracellular promastigotes. Treatment of the leishmaniases is far from ideal, and pentavalent antimonial (SbV) 1The abbreviations used are: SbV, pentavalent antimony; T[SH]2 and T[S]2, N1, N8-(bis)glutathionylspermidine (trypanothione) in the dithiol and disulfide forms, respectively; GspdSH, N1-glutathionylspermidine; SbIII, trivalent antimony; TCEP, tris(2-carboxyethyl)phosphine; MEM, Eagle's modified essential medium; HPLC, high pressure liquid chromatography; ovothiol, N1-methyl-4-mercaptohistidine. preparations such as sodium stibogluconate (Pentostam) have been the front-line drugs for more than half a century. However, the clinical value of antimony therapy is now threatened because of the emergence of drug resistance (2Sundar S. More D.K. Singh M.K. Singh V.P. Sharma S. Makharia A. Kumar P.C.K. Murray H.W. Clin. Infect. Dis. 2000; 31: 1104-1107Crossref PubMed Scopus (551) Google Scholar) and co-infection with human immunodeficiency virus (3Laguna F. Ann. Trop. Med. Parasitol. 2003; 97: 135-142Crossref PubMed Google Scholar). SbV is generally regarded as a pro-drug that first has to be activated by conversion to the trivalent form (SbIII) (4Goodwin L.G. Page J.E. Biochem. J. 1943; 37: 198-209Crossref PubMed Google Scholar). However, the site of reduction (host macrophage, amastigote, or both) and the mechanism of reduction (enzymatic or nonenzymatic) remain unclear (4Goodwin L.G. Page J.E. Biochem. J. 1943; 37: 198-209Crossref PubMed Google Scholar, 5Shaked-Mishan P. Ulrich N. Ephros M. Zilberstein D. J. Biol. Chem. 2001; 276: 3971-3976Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Frezard F. Demicheli C. Ferreira C.S. Costa M.A.P. Antimicrob. Agents Chemother. 2001; 45: 913-916Crossref PubMed Scopus (117) Google Scholar, 7Yan S. Li F. Ding K. Sun H. J. Biol. Chem. Inorg. Chem. 2003; 8: 689-697Crossref PubMed Scopus (93) Google Scholar, 8Denton H. McGregor J.C. Coombs G.H. Biochem. J. 2004; 381: 405-412Crossref PubMed Scopus (126) Google Scholar). The mode of action of these drugs is poorly understood. SbIII reversibly inhibits trypanothione reductase in vitro (9Cunningham M.L. Fairlamb A.H. Eur. J. Biochem. 1995; 230: 460-468Crossref PubMed Scopus (116) Google Scholar), but this has not been demonstrated in the intact parasite. However, inhibition of this unique and essential enzyme (10Tovar J. Cunningham M.L. Smith A.C. Croft S.L. Fairlamb A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5311-5316Crossref PubMed Scopus (165) Google Scholar, 11Tovar J. Wilkinson S. Mottram J.C. Fairlamb A.H. Mol. Microbiol. 1998; 29: 653-660Crossref PubMed Scopus (138) Google Scholar, 12Dumas C. Ouellette M. Tovar J. Cunningham M.L. Fairlamb A.H. Tamar S. Olivier M. Papadopoulou B. EMBO J. 1997; 16: 2590-2598Crossref PubMed Scopus (280) Google Scholar) is of particular interest, because trypanothione (N1,N8-bis(glutathionyl) spermidine (T[SH]2)) is a key intermediate in the regulation of thiol redox homeostasis, as well as in defense against chemical (13Vickers T.J. Fairlamb A.H. J. Biol. Chem. 2004; 279: 27246-27256Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Irsch T. Krauth-Siegel R.L. J. Biol. Chem. 2004; 279: 22209-22217Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and oxidative stress (15Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (691) Google Scholar, 16Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (184) Google Scholar). Paradoxically, more is known about the mechanism of resistance to SbIII than its mode of action (17Borst P. Ouellette M. Annu. Rev. Microbiol. 1995; 49: 427-460Crossref PubMed Scopus (268) Google Scholar). A considerable body of evidence implicates trypanothione and glutathione (γ-l-glutamyl-l-cysteinylglycine) in the efflux-mediated detoxification of SbIII and other heavy metals in the laboratory-induced resistant lines of Leishmania (18Dey 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, 19Mukhopadhyay R. Dey S. Xu N. Gage D. Lightbody J. Ouellette M. Rosen B.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10383-10387Crossref PubMed Scopus (215) Google Scholar). Overexpression of γ-glutamylcysteine synthetase (20Grondin K. Haimeur A. Mukhopadhyay R. Rosen B.P. Ouellette M. EMBO J. 1997; 16: 3057-3065Crossref PubMed Scopus (167) Google Scholar) and ornithine decarboxylase (21Haimeur A. Guimond C. Pilote S. Mukhopadhyay R. Rosen B.P. Poulin R. Ouellette M. Mol. Microbiol. 1999; 34: 726-735Crossref PubMed Scopus (116) Google Scholar) (the rate-limiting steps in the biosynthesis of the respective GSH and spermidine moieties of T[SH]2) and overexpression of PgpA (20Grondin K. Haimeur A. Mukhopadhyay R. Rosen B.P. Ouellette M. EMBO J. 1997; 16: 3057-3065Crossref PubMed Scopus (167) Google Scholar, 22Haimeur A. Brochu C. Genest P.A. Papadopoulou B. Ouellette M. Mol. Biochem. Parasitol. 2000; 108: 131-135Crossref PubMed Scopus (121) Google Scholar) (an intracellular metal-thiol transporter) have all been demonstrated to play a role in resistance. However, there are no reports implicating these effects in the mode of action of SbIII. Here we demonstrate that the mode of action of antimonials in drug-sensitive Leishmania is in fact intimately related to the mechanism of resistance. We also show that SbIII acts as a double-edged sword, not only does it decrease intracellular thiol buffer capacity by promoting loss of trypanothione and glutathione but it also increases the intracellular concentration of the disulfide forms of these thiols through inhibition of trypanothione reductase, thereby profoundly perturbing the thiol redox potential of the cell. Furthermore, we show for the first time that Pentostam, the clinically used pentavalent form, induces the same effects in axenically cultured amastigotes. Cell Lines and Culture Conditions—Leishmania donovani promastigotes (LV9 strain; WHO designation MHOM/ET/67/HU3) were propagated as described previously (23Ariyanayagam M.R. Fairlamb A.H. Mol. Biochem. Parasitol. 2001; 115: 189-198Crossref PubMed Scopus (173) Google Scholar). Cultures were initiated at 5 × 105 parasites per ml and grown at 24 °C with shaking. L. donovani amastigotes used in subsequent experiments were freshly isolated from the spleens of Golden hamsters (24Glaser T.A. Wells S.J. Spithill T.W. Pettitt J.M. Humphris D.C. Mukkada A.J. Exp. Parasitol. 1990; 71: 343-345Crossref PubMed Scopus (52) Google Scholar). Axenic amastigote cell line LdBOB (derived from MHOM/S.D./62/1S-CL2D) was maintained in culture as described previously (25Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar). To examine the effects of antimonial drugs on growth, triplicate cultures containing SbIII (as potassium antimony tartrate) or SbV (as sodium stibogluconate, free of m-chlorocresol, a gift from GlaxoSmith-Kline) were seeded with 5 × 105 parasites/ml. Cell densities were determined microscopically after culture for 72 h, and IC50 values were determined using the IC50 4-parameter equation provided with GraFit. Analysis of Intracellular Thiols—Mid-log promastigotes were centrifuged (1,600 × g, 10 min, 4 °C), resuspended in fresh culture medium at 1 × 107 ml-1, and incubated with antimony. The viability of cells was monitored microscopically and by the LIVE/DEAD™ Viability/Cytotoxicity assay (Molecular Probes). At intervals, 5 × 107 promastigotes were collected by centrifugation and derivatized with monobromobimane as described previously (26Shim H. Fairlamb A.H. J. Gen. Microbiol. 1988; 134: 807-817PubMed Google Scholar). In experiments where amastigotes were analyzed, 5 × 108 freshly harvested parasites, or 1 × 108 axenic amastigotes, were used. 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 fluorometer. Recovery of Reduced Thiol Levels Following Removal of SbIII—Midlog promastigotes were incubated in either Grace's medium or in a minimal maintenance medium (44 mm NaCl, 56 mm glucose, 56 mm Na2HPO4, 3 mm NaH2PO4, pH 8) containing 112 μg ml-1 SbIII and at a cell density of 1 × 107 ml-1. Following a 2.5-h incubation, cells were washed once with fresh medium and resuspended in either Grace's or minimal media at 1 × 107 ml-1. At intervals throughout the 2.5-h SbIII incubation and for a further 2.5 h following removal of SbIII, aliquots of culture were analyzed for thiols. Regeneration of Reduced Intracellular Thiols—Diamide (diazine dicarboxylic acid bis(N,N-dimethylamide)), from Sigma, was used to oxidize intracellular thiols as described previously (27Kelly J.M. Taylor M.C. Smith K. Hunter K.J. Fairlamb A.H. Eur. J. Biochem. 1993; 218: 29-37Crossref PubMed Scopus (76) Google Scholar). Cultures were preincubated at 107 ml-1 at 24 °C for 1 h in fresh medium (controls) or in medium containing 112 μg ml-1 SbIII. Cells were centrifuged (1,600 × g, 10 min, 4 °C) and resuspended at 3 × 107 ml-1 in ice-cold maintenance buffer containing 5 mm diamide and SbIII. Following incubation (20 min, 4 °C), cells were pelleted by centrifugation and resuspended at 5 × 107 ml-1 in fresh culture medium with or without SbIII. The viability of cells following treatment with diamide was monitored as described above. At intervals, aliquots were removed and analyzed for thiol content as described above. Amastigotes were treated in an identical manner. Radiolabeling of L. donovani Promastigotes—Mid-log promastigotes were pelleted by centrifugation and resuspended at 2 × 107 ml-1 in Eagle's modified essential medium (MEM) without l-glutamine and methionine. l-[35S]Methionine was subsequently added to the cultures to 1 μCi ml-1 and incubated at 24 °C for ∼12 h. Following incubation, cells were washed twice with fresh, unlabeled MEM at 4 °C and chased for 30 min in the absence of label. Following one further wash at 4 °C, labeled promastigotes were resuspended in fully supplemented MEM at 5 × 107 ml-1 in the presence and absence of SbIII (112 μg ml-1). At defined intervals, 1-ml aliquots of culture were pelleted by centrifugation, and the supernatant was removed. Supernatants (0.5 ml) were mixed with 5 ml of scintillation fluid (Pico-Fluor 40) and counted in an LS 6000 LL scintillation counter (Beckman Instruments). Radiolabeled thiols in these supernatants were identified by HPLC as above, only in this instance, each sample was reduced using a 4-5 molar excess of tris(2-carboxyethyl)phosphine (TCEP) prior to derivatization. Fractions were collected at 1-min intervals, and radioactivity was determined. Analysis of Oxidized Intracellular Thiols—At 0, 1.5, and 3 h following incubation with SbIII (112 μg ml-1), duplicate sets of 5 × 107 promastigotes were harvested. In one set, the reduced thiol levels were determined, and in the other set the total thiol content was determined by TCEP reduction, prior to derivatization. Levels of disulfide within these parasites were calculated by comparing levels of reduced thiol, with the total thiol content determined following reduction with TCEP. Amastigotes were treated in an identical manner. Axenic amastigotes were treated with SbV (600 μg ml-1) over a 24-h period. Electron potentials for GSSG/2GSH (-240 mV) (28Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar) and T[S]2/T[SH]2 (-242 mV) (29Fairlamb A.H. Henderson G.B. NATO ASI Series Series H Cell Biol. 1987; 11: 29-40Google Scholar) were calculated assuming an intracellular pH of 7.0 and using the appropriate Nernst equation (28Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). For GSSG/2GSH (in mV at 25 °C, pH 7.0) (Equation 1), E=E0′−(59.1n)log([GSH]2[GSSG])(Eq. 1) For T[S]2/T[SH]2 (in mV at 25 °C, pH 7.0) (Equation 2), E=E0′−(59.1n)log([T(SH)2][T(S)2])(Eq. 2) The cell volumes used in this calculation were measured using a Schärfe Systems CASY1 cell counter yielding 2.86 ± 0.02 and 0.97 ± 0.01 μl (108 cells)-1 for L. donovani promastigotes and amastigotes, respectively, and 1.74 ± 0.04 μl (108 cells)-1 for LdBOB amastigotes. The Effects of Trivalent Antimony on the Intracellular Thiol Levels of L. donovani Promastigotes—The time-dependent effects of a fixed concentration of SbIII on promastigote intracellular thiol levels were examined in detail (Fig. 1). In the absence of SbIII, levels of trypanothione, glutathione, and GspdSH remained stable throughout a 4-h time course (Fig. 1A, open symbols). However, treatment of promastigotes with potassium antimony tartrate (SbIII concentration of 112 μg ml-1, three times the IC50 value) resulted in a significant, time-dependent decrease in the levels of all glutathione-containing thiols (Fig. 1A, closed symbols). The overall disappearance of these thiols is linear (76 ± 5 pmol min-1 (108 cells)-1, R = 0.99, this experiment; 60 ± 2 pmol min-1 (108 cells)-1, R = 0.99, duplicate experiment), decreasing to ∼15% of their initial value after a 4-h exposure to SbIII (Fig. 1B, closed squares). In contrast, the levels of ovothiol (N1-methyl-4-mercaptohistidine) within promastigotes were unaffected by potassium antimony tartrate (Fig. 1B, triangles), indicating that loss of thiols because of cell lysis is not responsible for these effects. This was confirmed by phase contrast microscopy, which showed the parasites to be fully motile although somewhat more rounded than untreated controls, and by a fluorescent cell viability assay (not shown). Similar effects on thiol levels were observed with amastigotes isolated from the spleens of infected hamsters (see below), except that ovothiol is not detectable in this life cycle stage as reported previously (23Ariyanayagam M.R. Fairlamb A.H. Mol. Biochem. Parasitol. 2001; 115: 189-198Crossref PubMed Scopus (173) Google Scholar). The disappearance of T[SH]2 was more rapid and pronounced in promastigotes treated with SbIII in a minimal medium, lacking cysteine, methionine, or GSH, than in fully supplemented culture medium (Fig. 1C). Removal of SbIII from supplemented cultures after 2.5 h resulted in the rapid recovery of T[SH]2 to 89% of initial levels within a further 2.5 h, demonstrating the reversibility of SbIII-induced thiol loss. However, promastigotes in a nonsupplemented minimal medium failed to recover from prior exposure to potassium antimony tartrate, reaching only 35% of initial levels, suggesting that recovery largely depends on the ability of the parasites to re-synthesize lost thiols. The effects of a range of SbIII concentrations on the levels of intracellular thiols were measured following 4 h of exposure (Fig. 1D). The loss of GSH-containing species is dose-dependent (IC50 10.5 ± 2.0 μg ml-1) before saturating at SbIII concentrations nearing 112 μg ml-1 (Fig. 1D, closed symbols). As expected, levels of ovothiol remained comparatively constant despite increasing concentrations of SbIII (Fig. 1D, open symbols). Antimony-induced Thiol Efflux from L. donovani Promastigotes—The fate of cellular thiols following treatment with SbIII was monitored in culture supernatants of promastigotes radiolabeled previously with [35S]methionine (Fig. 2, A and B). Following reduction with TCEP and derivatization with monobromobimane, medium supernatants were analyzed by HPLC, and the elution times of radiolabeled peaks were compared with known standards. Fig. 2A shows specimen data of cell-free medium obtained following 4 h of incubation in the presence and absence of SbIII. Major peaks of radioactivity, which coeluted with the bimane derivatives of T[SH]2 and GSH standards, were observed in SbIII-treated samples, whereas these peaks were not seen in the untreated control. In contrast, a major radioactive peak corresponding to [35S]methionine was evident in both SbIII-treated and control supernatants presumably as a result of exchange with unlabeled methionine in the culture medium. It is apparent from these studies that incubation of promastigotes with SbIII results in a very specific, time-dependent efflux of glutathione-containing thiols. The absence of other labeled thiol species such as ovothiol in supernatants suggests that no significant cell lysis has taken place and that efflux is limited to GSH and T[SH]2. Efflux of labeled GSH and T[SH]2 is linear for about 2 h but decreases slightly thereafter, possibly because of dilution of the radiolabeled pool by de novo synthesis of unlabeled GSH (Fig. 2B). The mean ratio of radioactivity in T[SH]2 and GSH for each time point is 1.8 ± 0.2. Since T[SH]2 contains two GSH moieties, this indicates that approximately equimolar amounts of T[SH]2 and GSH are effluxed in response to SbIII treatment. Inhibition of Thiol Regeneration by Trivalent Antimony in L. donovani Promastigotes and ex Vivo Amastigotes—Previous studies (9Cunningham M.L. Fairlamb A.H. Eur. J. Biochem. 1995; 230: 460-468Crossref PubMed Scopus (116) Google Scholar) undertaken in this laboratory have demonstrated that SbIII inhibits trypanothione reductase in a time-dependent and reversible manner in vitro. Unfortunately, direct demonstration that trypanothione reductase is inhibited in intact cells proved impossible, because the enzyme-inhibitor complex rapidly dissociates on dilution following cell extraction and assay (9Cunningham M.L. Fairlamb A.H. Eur. J. Biochem. 1995; 230: 460-468Crossref PubMed Scopus (116) Google Scholar). However, we have shown previously that the ability of cells to regenerate thiols from the disulfide forms, following transient exposure to the oxidant diamide, is a suitable indirect method of assessing trypanothione reductase activity within intact cells (10Tovar J. Cunningham M.L. Smith A.C. Croft S.L. Fairlamb A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5311-5316Crossref PubMed Scopus (165) Google Scholar, 27Kelly J.M. Taylor M.C. Smith K. Hunter K.J. Fairlamb A.H. Eur. J. Biochem. 1993; 218: 29-37Crossref PubMed Scopus (76) Google Scholar). By using this method, we have examined the effect of SbIII on thiol recovery in L. donovani (Fig. 3). Immediately following diamide treatment, T[SH]2 and GSH levels were below the limits of detection. After resuspension of cells into fresh minimal medium, in the absence of SbIII, promastigotes were able to regenerate rapidly T[SH]2 and GSH to between 60 and 80% of control values within 5 min (Fig. 3, A and B, respectively). In contrast, in cells exposed to SbIII throughout the experiment, both the rate of recovery and the final thiol level were significantly impaired. An even more pronounced effect was observed with the clinically relevant amastigote stage of the parasite (Fig. 3, C and D, respectively). The initial rates of regeneration of T[SH]2 and GSH were ∼5-6-fold slower than in control cells indicating that trypanothione reductase activity was inhibited by >80%. Effect of Trivalent Antimony on Levels of Disulfide within L. donovani Promastigotes and ex Vivo Amastigotes—If SbIII inhibits trypanothione reductase in intact cells, then the levels of trypanothione and glutathione disulfide might be expected to rise in the face of either endogenous or exogenous oxidant stress as well as other thiol-dependent metabolic processes. As pre-column derivatization of whole cells with monobromobimane merely detects free thiols, the total thiol content was determined by reducing any disulfides prior to derivatization using the reducing agent TCEP. The difference in thiol content in the presence and absence of TCEP thus represents the disulfide content. In promastigotes and amastigotes incubated in the absence of SbIII, 96 and 98% of the total trypanothione and glutathione pools were recovered as the free thiol (Fig. 4, A and D, respectively). Note, however, that amastigotes contain ∼5-fold lower concentrations of both thiols than promastigotes. After a 3-h exposure to SbIII, the free thiol content in both cell types decreased by 70-80%, in agreement with results shown in Fig. 1. This decline in T[SH]2 and GSH was associated with an increase in trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG) such that after 3 h they accounted for about 50% of total trypanothione and glutathione in promastigotes and about 40% in amastigotes (Fig. 4, B and E, respectively). Absolute concentrations of thiols and disulfides within SbIII-treated promastigotes and amastigotes were used to calculate half-cell reduction potentials for the T[S]2/T[SH]2 and GSSG/2GSH couples at pH 7.0 (Fig. 4, C and F) (28Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). Both promastigotes and amastigotes maintain intracellular pH values close to neutral, independent of external pH (30Glaser T.A. Baatz J.E. Kreishman G.P. Mukkada A.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7602-7606Crossref PubMed Scopus (74) Google Scholar). In both developmental forms of L. donovani, the reduction potential for the trypanothione couple was lower than the glutathione couple, indicating that the two systems are apparently not at equilibrium. A similar situation has been noted for the equilibrium between glutathione, which forms intermolecular disulfides, and thioredoxin, which forms intramolecular disulfides (28Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). This is because the redox potential for glutathione is concentration-dependent, whereas that of thioredoxin is not (28Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3676) Google Scholar). After exposure to SbIII for 3 h, the redox potentials for both couples became markedly less electro-negative (i.e. indicative of a more oxidizing environment) with the reduction potential of the GSSG/2GSH couple increasing by +61 and +60 mV and the T[S]2/T[SH]2 couple increasing by +36 and +46 mV in promastigotes and amastigotes, respectively. Effects of Pentavalent Antimony on the Thiol Metabolism of LdBOB Promastigotes and Axenic Amastigotes—It is generally accepted that pentavalent antimonials, such as Glucantime and Pentostam, act as prodrugs requiring reduction to SbIII before becoming biologically active. Controversy surrounds the mechanism of SbV bio-activation, and it remains unclear whether the amastigote stage of the parasite or the macrophage host cell is responsible. To address this question, a well characterized axenic amastigote cell line (LdBOB) was used to investigate the effects of SbV on intracellular thiol levels of isolated amastigotes (25Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar). Initially, the sensitivity of LdBOB amastigotes and promastigotes for Sb was established by determining IC50 values for both SbIII and SbV (Fig. 5A). In keeping with published data (25Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (147) Google Scholar), we obtained IC50 values of 8 and 11 μg ml-1 for SbIII in LdBOB amastigotes and promastigotes, respectively. SbV was selectively toxic for the amastigote stage of Leishmania with an IC50 of 200 μg ml-1, whereas LdBOB promastigotes were totally insensitive to SbV concentrations up to 1000 μg ml-1. To assess the effects of SbV on intracellular thiols, mid-log axenic amastigotes were incubated with 600 μg ml-1 SbV (3× IC50), and thiols were analyzed over a 24-h period. The levels of both T[SH]2 and GSH fell steadily throughout the incubation with SbV (Fig. 5B), with overall levels of GSH-containing species falling by ∼90% (∼4 pmol min-1 (108 cells)-1, Fig. 5C). The effects of SbV on thiol redox balance in LdBOB amastigotes were monitored over 24 h. In addition to the loss of GSH-containing thiols, incubation with SbV was accompanied by a gradual accumulation of disulfide within LdBOB amastigotes such that after 24 h trypanothione and glutathione disulfide accounted for 48 and 49% of the total (Fig. 6, A and B). In addition, exposure of LdBOB amastigotes to SbV also leads to a significant alteration in the redox potentials of both T[SH]2 and GSH (Fig. 6C). In keeping with our previous observations with SbIII (see Fig. 4F), the redox potentials of GSH and T[SH]2 became markedly less electro-negative throughout 24 h, increasing by +63 and +37 mV for GSH and T[SH]2, respectively. Despite almost a century of use, virtually nothing is known about the mode of action of antimonial drugs. Early work suggested that Pentostam (SbV) inhibits energy metabolism and macromolecular biosynthesis via inhibition of glycolysis and fatty acid β-oxidation (31Berman J.D. Waddel D. Hanson B.D. Antimicrob. Agents Chemother. 1985; 27: 916-920Crossref PubMed Scopus (108) Google Scholar, 32Berman J.D. Gallalee J.V. Best J.M. Biochem. Pharmacol. 1987; 36: 197-201Crossref PubMed Scopus (91) Google Scholar). The specific targets have not yet been identified, but hexokinase, phosphofructokinase, and pyruvate kinase have been eliminated as possible candidates (33Mottram J.C. Coombs G.H. Exp. Parasitol. 1985; 59: 151-160Crossref PubMed Scopus (79) Google Scholar). SbIII also inhibits trypanothione reductase in vitro, but the significance of this observation until now has remained obscure (9Cunningham M.L. Fairlamb A.H. Eur. J. Biochem. 1995; 230: 460-468Crossref PubMed Scopus (116) Google Scholar). More recent studies (34Sereno D. Holzmuller P. Mangot I. Cuny G. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 2001; 45: 2064-2069Crossref PubMed Scopus (125) Google Scholar, 35Sudhandiran G. Shaha C. J. Biol. Chem. 2003; 278: 25120-25132Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) have reported that SbIII-treated amastigotes undergo death by an apoptotic-like mechanism involving DNA fragmentation and externalization of phosphatidylserine by a caspase-independent mechanism. Three key observations arising from this study are relevant to the mode of action of antimonials against drug-sensitive L. donovani (Fig. 7). First, SbIII induces efflux of trypanothione and glutathione from both stages of the life cycle. Second, significant amounts of both thiols accumulate inside cells as their respective disulfides. The net result of these two effects is to significantly reduce intracellular thiol-buffering capacity and profoundly affect the thiol redox potential. Third, SbV (as Pentostam) induces similar effects in the mammalian (amastigote) form but not the insect (promastigote) form of the parasite. The latter observa
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