Functional Involvement of Leishmania donovani Tryparedoxin Peroxidases during Infection and Drug Treatment.

The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.

The antiparasitic drug, potassium antimony tartrate, inhibits tumor angiogenesis and tumor growth in nonsmall-cell lung cancer.

Repurposing existing drugs not only accelerates drug discovery but rapidly advances clinical therapeutic strategies. In this article, we identified potassium antimonyl tartrate (PAT), an antiparasitic drug, as a novel agent to block angiogenesis by screening US Food and Drug Administration-approved chemical drugs. By comparing the cytotoxicity of PAT in various nonsmall-cell lung cancer (NSCLC) cells with that observed in primary cultured human umbilical vein endothelial cells (HUVECs), we found that HUVECs were much more sensitive to the PAT treatment. In in vivo tumor xenograft mouse models established either by PAT-resistant A549 cells or by patient primary tumors, PAT significantly decreased the tumor volume and tumor weight of NSCLC xenografts at dosage of 40 mg/kg (i.p., daily) and, more importantly, augmented the antitumor efficacy of cisplatin chemotherapy. Remarkable loss of vascularization in the treated xenografts indicated the in vivo antiangiogenesis property of PAT, which was well correlated with its tumor growth inhibition in NSCLC cells. Furthermore, in the in vitro angiogenic assays, PAT exhibited dose-dependent inhibition of HUVEC proliferation, migration, and tube formation in response to different stimuli. Consistently, PAT also abolished the vascular endothelial cell growth factor-induced angiogenesis in the Matrigel plugs assay. Mechanistically, we found that PAT inhibited the activities of several receptor tyrosine kinases and specifically blocked the activation of downstream Src and focal adhesion kinases in HUVECs. Taken together, our results characterized the novel antiangiogenic and antitumor function of PAT in NSCLC cells. Further study of PAT in anticancer clinical trials may be warranted.

Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis.

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 µM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 µM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

Selection and phenotype characterization of potassium antimony tartrate-resistant populations of four New World Leishmania species.

In the present study, we selected in vitro populations of Leishmania Viannia guyanensis, L.V. braziliensis, L. Leishmania amazonensis and L.L. infantum chagasi that were resistant to potassium antimony tartrate (SbIII). The resistance index of these populations varied from 4- to 20-fold higher than that of their wild-type counterparts. To evaluate the stability of the resistance phenotype, these four resistant populations were passaged 37 to 47 times in a culture medium without SbIII. No change was observed in the resistance indexes of L.V. guyanensis (19-fold) and L.L. infantum chagasi (4-fold). In contrast, a decrease in the resistance index was observed for L.V. braziliensis (from 20- to 10-fold) and L.L. amazonensis (from 6- to 3-fold). None of the antimony-resistant populations exhibited cross-resistance to amphotericin B and miltefosine. However, the resistant populations of L.V. braziliensis, L.L. amazonensis and L.L. infantum chagasi were also resistant to paromomycin. A drastic reduction was observed in the infectivity in mice for the resistant L.V. guyanensis, L.L. amazonensis and L.V. braziliensis populations. The SbIII-resistant phenotype of L.V. braziliensis was stable after one passage in mice. Although the protocol of induction was the same, the SbIII-resistant populations showed different degrees of tolerance, stability, infectivity in mice and cross-resistance to antileishmanial drugs, depending on the Leishmania species.

Comprehensive investigations on anti-leishmanial potentials of Euphorbia wallichii root extract and its effects on membrane permeability and apoptosis.

Clinically available synthetic chemotherapeutics to treat the vector-borne protozoan infection, leishmaniasis, are associated with serious complications such as toxicity and emergence of resistance. Natural products from plants consist of interesting biomolecules that may interfere with DNA or membrane integrity of the parasite and can possibly minimise the associated side effects. In the present study, various fractions of Euphorbia wallichii (EW) root extracts including n-hexane (EWNX), ethyl acetate (EWEA), chloroform (EWCH) and aqueous (EWAQ), were evaluated for their antileishmanial potential against Leishmania tropica followed by investigation of the possible mechanism of action via reactive oxygen species (ROS) quantification, membrane permeability (via sytox green dye) and apoptotic assay (via AO/EB method) using fluorescent microscopy. Two of the fractions i.e. EWEA and EWAQ inhibited the growth of promastigotes (IC50 7.8 and 10.2 µg/mL, respectively) and amastigotes (IC50 9.9 and 13.3 µg/mL, respectively) forms almost at similar concentrations as found for the standard antileishmanial drugs, tartar emetic (TA) and Glucantime (IC50 9.4 and 21.5 µg/mL, respectively). Both the active fractions remained non-toxic towards human blood erythrocytes and were able to cause membrane permeability and apoptotic induction (using Triton X-100 as a positive control) leading to death of Leishmania parasites. However, both the fractions could not triger significant and persistent ROS generation, compared to hydrogen peroxide used as a positive control. Antilesihmanial activity of the two active fractions might be attributed to the presence of high quantity of tannins and saponins.

A protein of the leucine-rich repeats (LRRs) superfamily is implicated in antimony resistance in Leishmania infantum amastigotes.

Pentavalent antimonial containing drugs (SbV) are the mainstay for the control of the protozoan parasite Leishmania but resistance to this class of drug is now prevalent in several endemic areas. We describe here the use of functional cloning where an expression cosmid bank derived from Leishmania infantum was transfected in L. infantum axenic amastigotes and selected for potassium antimonyl tartrate (SbIII) resistance. This strategy allowed the isolation of a cosmid encoding for a novel resistance protein, LinJ34.0570, which belongs to the superfamily of leucine-rich repeat (LRR) proteins. Parasites overexpressing this LRR protein, which is part of the LRR_CC subfamily, were resistant to SbIII as axenic amastigotes and to SbV as intracellular parasites. This work pinpoints a novel protein that can contribute to antimonial resistance in Leishmania.

Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent.

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

Identification of cisplatin sensitizers through high-throughput combinatorial screening.

cis-Diamminedichloroplatinum/cisplatin (CDDP) is a major drug used in cancer chemotherapy; however, the toxic side-effects and development of drug resistance represent major challenges to the clinical use of CDDP. The aim of the present study was to identify effective drug combination regimens through high-throughput drug screening that can enhance the efficacy of CDDP, and to investigate the underlying mechanisms. A cell-based high-throughput screening methodology was implemented, using a library of 1,280 Food and Drug Administration (FDA)-approved drugs, to identify clinical compounds that act synergistically with CDDP. Our study identified two compounds, namely potassium antimony tartrate and topotecan, that significantly enhanced the sensitivity of colorectal and non-small cell lung cancer cells to CDDP. The synergistic action of both compounds with CDDP was confirmed by further quantitative analyses. Topotecan is a topoisomerase-1 inhibitor that has previously been shown to enhance the clinical response and overall patient survival when combined with CDDP by a yet unclear mechanism. We demonstrated that the combination of topotecan with CDDP significantly inhibited colony formation ability and increased the apoptosis of several cancer cell lines. Mechanistic analyses revealed that topotecan enhanced CDDP-induced DNA damage and inhibited the repair of DNA strand breaks, without affecting the cellular platinum content. Overall, the findings of this study demonstrated that the use of the FDA-approved drug panel in high-throughput screening is an effective method for identifying effective therapeutic regimens that are clinically relevant, and may have high feasibility for translation into clinical practice.

Cross-resistance between cisplatin, antimony potassium tartrate, and arsenite in human tumor cells.

Cross-resistance between cisplatin (DDP) and metalloid salts in human cells was sought on the basis that mechanisms that mediate metalloid salt cross-resistance in prokaryotes are evolutionarily conserved. Two ovarian and two head and neck carcinoma cell lines selected for DDP resistance were found to be cross-resistant to antimony potassium tartrate, which contains trivalent antimony. The DDP-resistant variant 2008/A was also cross-resistant to arsenite but not to stibogluconate, which contains pentavalent antimony. A variant selected for resistance to antimony potassium tartrate was cross-resistant to DDP and arsenite. Resistance to antimony potassium tartrate and arsenite was of a similar magnitude (3-7-fold), whereas the level of resistance to DDP was greater (17-fold), irrespective of whether the cells were selected by exposure to DDP or to antimony potassium tartrate. In the resistant sublines, uptake of [3H]-dichloro(ethylenediamine) platinum(II) was reduced to 41-52% of control, and a similar deficit was observed in the accumulation of arsenite. We conclude that DDP, antimony potassium tartrate, and arsenite all share a common mechanism of resistance in human cells and that this is due in part to an accumulation defect.

Differential toxicity of antimonial compounds and their effects on glutathione homeostasis in a human leukaemia monocyte cell line.

Trivalent antimonial compounds (Sb(III)), originally used in the treatment of leishmaniasis, are now being proposed as a novel therapy for acute promyelocytic leukaemia (APL). Here, we examine the effects of Sb(III) and pentavalent antimonial drugs (Sb(V)) on glutathione homeostasis, oxidative stress and apoptosis in the human leukaemia monocyte cell line, THP-1. Although growth of THP-1 macrophages is unaffected by Sb(V), macrophages are extremely sensitive to Sb(III). On exposure to Sb(III), intracellular free glutathione (GSH) levels in macrophages decrease linearly by 50% over 4h, associated with efflux of both GSH and accumulation of intracellular glutathione disulphide (GSSG). Together these effects increase the redox potential of the GSSG/GSH couple from -282 to -225mV. Sb(III)-induced GSH efflux from THP-1 macrophages is accompanied by the concomitant efflux of Sb(III) at a constant molar ratio of 3 (GSH) to 1 (Sb(III)), respectively. Sb(III) directly inhibits glutathione reductase activity in macrophages, significantly retarding the regeneration of GSH from GSSG, following diamide oxidation. Sb(III)-treated THP-1 macrophages go on to exhibit elevated levels of reactive oxygen species and show the early signs of apoptosis. The absence of these effects in Sb(V)-treated THP-1 cells suggests that macrophages do not efficiently reduce Sb(V) to Sb(III). Collectively, these findings suggest that Sb(III) seriously compromises thiol homeostasis in THP-1 macrophages and that this may be an early defining event in the mode of action of antimonials against leukaemia cells.

Purification, kinetics and inhibition by antimonials of recombinant phosphofructokinase from Schistosoma mansoni.

We reported before on the cloning of a cDNA encoding S. mansoni PFK. In the present investigation we established optimal conditions for expression of the enzyme in insect cells with high yield. The recombinant PFK was purified to homogeneity. Kinetic properties of the pure enzyme were studied with respect to its two substrates, Fru-6-P and ATP, and were compared with properties of mammalian PFK. ATP inhibited the parasite enzyme only at concentrations higher than those which inhibited mammalian muscle PFK. Saturation curves for Fru-6-P showed typical cooperative kinetics. AMP, cAMP and Fru-2,6-bisP activated the enzyme causing reduced apparent Km for Fru-6-P and an increase in maximal activity. Both ATP inhibition and cooperative kinetics for Fru-6-P occur at both pH 6.9 and 8.2. This is a distinct difference from the mammalian enzyme which shows these kinetic properties only at neutral or slightly acidic pH, but not at an alkaline pH. Recombinant PFK is more sensitive to inhibition by the trivalent antimonials, antimony potassium tartrate and Stibophen, than is the mammalian heart muscle enzyme. The inhibition is at least partially antagonized by the sulfhydryl protective reagent, dithiothreitol.

Changes in glucose metabolism and cyanide sensitivity in Schistosoma mansoni during development.

Schistosoma mansoni was studied by biochemical and electrophysiological techniques to follow the physiological changes occurring during transformation in the mammalian host. Volume conducted electrical potentials and measurement of CO2 evolution indicate that 3 h post-transformational schistosomula are highly sensitive to cyanide. By 24 h after transformation, evolution of CO2 under control conditions is reduced by 77% from 3 h levels, while lactate excretion rises by 84%. Cyanide does not affect the frequency or magnitude of endogenous electrical transients, but does eliminate 83% of the already reduced levels of CO2 evolved in 24 schistosomula. Electrophysiological analyses indicate that the timecourse of metabolic changes in skin- and mechanically transformed schistosomula are similar, and incubation of schistosomula in 200 micrograms ml-1 puromycin does not alter the onset of cyanide insensitivity. The adult parasite evolves a low level of CO2 which is reduced by 88% in the presence of 1 mM cyanide. No significant Pasteur effect is detected, however, and endogenous electrical activity as well as mechanical responses of the adult musculature are unaffected by cyanide exposure. Our results indicate that schistosomula continue to rely on cyanide-sensitive respiratory components for at least 3 h after transformation; by 24 h, however, the parasites are metabolically similar to the adult stage, i.e., they depend on lactate fermentation for most of their energy requirements.

Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth.

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania.

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.

Potassium antimonyl tartrate induces reactive oxygen species-related apoptosis in human myeloid leukemic HL60 cells.

Potassium antimonyl tartrate (PAT), like arsenic trioxide (As2O3), has recently been shown to exert cytotoxicity towards acute promyelocytic leukemia (APL) cells. In the present study, we demonstrated that PAT treatment also inhibited cell growth of four acute myeloid leukemia (AML) cell lines, i.e., HL60, K562, KG1a and U937, that do not derive from APL. PAT, like As2O3, was further shown to induce apoptosis in HL60 cells as assessed by Hoechst 33342 staining and microscopical detection. Such an apoptotic process was associated with loss of mitochondrial potential and enhanced cellular production of reactive oxygen-related species; it was potentiated by co-treatment with buthionine sulfoximine, a pro-oxidant compound acting through inhibition of glutathione synthesis, and abolished in response to the antioxidant N-acetylcysteine, thus outlining that the toxicity of PAT, similarly to that of As2O3, is modulated by the cellular redox status. Pan-caspase inhibitors failed to inhibit PAT-triggered apoptosis of HL60 cells whereas they fully blocked that linked to As2O3, suggesting that PAT, unlike As2O3, does not require caspase activity for inducing apoptosis. PAT and As2O3 also differently affected intracellular pH, a key parameter commonly altered during apoptotic processes. Such data therefore indicate that PAT can exert cytotoxicity towards AML cells not deriving from APL such as HL60 cells, through inducing an apoptotic process which exhibits some similarities and some differences with that triggered by As2O3.

Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

Inhibition of DNA-double strand break repair by antimony compounds.

DNA double strand breaks (DSBs), induced by gamma-irradiation in Chinese hamster ovary cells, were used to examine whether antimony compounds affect the repair of DNA damage. The cells were first incubated with antimony trichloride or antimony potassium tartrate (both Sb(III)) for 2 h, and then irradiated with gamma-rays at a dose of 40 Gy. The DNA DSB was quantified with pulsed field gel electrophoresis immediately after irradiation (non-repair group) as well as at 30 min post-irradiation (repair group). The degree of repair inhibition was determined by the differences in the amount of DNA DSB between non-repair and repair groups. Both antimony compounds inhibited repair of DNA DSB in a dose dependent manner. In trichloride, 0.2 mM antimony significantly inhibited the rejoining of DSB, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses, D(0), for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D(0), but the antimony potassium tartrate inhibited the repair at doses where most cells lost their proliferating ability. This is the first report to indicate that antimony compounds may inhibit the repair of radiation-induced DNA DSB.

The effects of stibophen on phosphofructokinases and aldolases of adult filariids.

Trivalent organic antimonials, such as stibophen, have been employed for the chemotherapy of schistosome and filariid infections. The effects of stibophen on adult Litomosoides carinii, Dipetalonema witei (= viteae), and Brugia pahangi were examined. In vitro, lactate accumulation was markedly inhibited by the antimonials as was phosphofructokinase activities in homogenates. Incubation of filariids with stibophen and determination of internal concentrations of hexose phosphate also indicated a decreased phosphofructokinase activity. In addition, a second inhibitory effect of stibophen on aldolase has been observed which appears to be specific for stibophen and is not displayed by potassium antimony tartrate. Both inhibitory activities may contribute to the chemotherapeutic effect of stibophen. In addition to the schistosomes and filariids, stibophen also inhibits Ascaris and Hymenolepis diminuta phosphofructokinases at low concentrations, where no inhibition of the corresponding mammalian liver enzyme was demonstrable.

Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents.

We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P < 0.05) 3-fold greater potency against the L. donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives.

In vitro susceptibility to antimonials and amphotericin B of Leishmania infantum strains isolated from dogs in a region lacking drug selection pressure.

The aim of this study was to evaluate the susceptibility to anti-leishmanial agents of 24 strains isolated from dogs living in the urban area of Alger lacking drug selection pressure. Two different Leishmania infantum zymodemes, MON-1 and MON-281, were identified in these dogs. The in vitro susceptibility to the main forms of antimonial and amphotericin were assessed on promastigote and amastigote life stages in culture. The results obtained for both parasite life stages were concordant whatever the molecule tested. Moreover, our data showed that isolates belonging to the relatively rare zymodeme of L. infantum, MON-281, were less susceptible to antimony than MON-1, when at the same time there was no significant difference for amphotericin B.