Glutathione-Induced Conversion of Pentavalent Antimony to Trivalent Antimony in Meglumine Antimoniate

Abstract

The standard treatment of human leishmaniases involves the use of pentavalent antimony [Sb(V)] compounds, including meglumine antimoniate. The mode of action of these compounds has not been fully elucidated. The possibility that Sb(III) is involved has been suggested; however, the biomolecule that may induce the conversion of Sb(V) to Sb(III) has not yet been identified. In the present study, we investigated both the ability of reduced glutathione (GSH) to promote the reduction of Sb(V) into Sb(III) in meglumine antimoniate and the effects of pH and temperature on this transformation. GSH did promote the reduction of Sb(V) into Sb(III) in a dose-dependent manner. When GSH and meglumine antimoniate were incubated together at a GSH/Sb molar ratio superior or equal to 5:1, all antimony was encountered in the reduced form, indicating a stoichiometry of 5:1 between GSH and Sb(V) in the reaction. The reaction between Sb(V) and GSH was favored at an acidic pH (pH 5) and an elevated temperature (37°C), conditions found within the phagolysosome, in which Leishmania resides. For instance, about 30% of the Sb(V) (concentration, 2mM) was converted to Sb(III) following incubation for 3 days with 10 mM GSH at pH 5 and 37°C. Our data support the hypothesis that Sb(V) would be converted by GSH, or a related thiol compound, to more toxic Sb(III) in the phagolysosome of macrophages.The leishmaniases are a group of diseases produced by invasion of the reticuloendothelial system of a vertebrate host by a parasite of the genus Leishmania. This parasite is found as a motile promastigote in the sandfly; it transforms into an amastigote when engulfed by host macrophages and resides in the acidic environment of secondary lysosomes (1). These diseases are a significant cause of morbidity and mortality in several countries of the world. The treatment of choice for all forms of leishmaniasis depends on pentavalent antimony [Sb(V)]-containing drugs such as meglumine antimoniate (Glucantime) and sodium stibogluconate (Pentostam). Despite the clinical use of these antileishmanial agents for over half a century, their mechanism of action and the basis for their selective toxicity remain unknown. The hypothesis that Sb(V) acts as a prodrug that is converted to the more toxic trivalent antimony [Sb(III)] at or near the site of action was first suggested by Goodwin and Page (9), after they observed that a host organism can reduce Sb(V) into Sb(III). Recently, hydride generation-atomic absorption spectrometry analysis of serum and urine from patients treated with meglumine antimoniate revealed that 15 to 25% of serum antimony and 50% of urine antimony were trivalent (3, 15). This hypothesis was further supported by the observations that Sb(III) is more toxic than Sb(V) against both parasite stages of different Leishmania species (13,19,21) and that mutants of Leishmania infantum amastigotes selected for resistance to Sb(III) were cross-resistant to Sb(V) inside monocytes (22).Recently, however, Ephros et al. (6) showed that axenically grown amasti...

Drug Resistance in Leishmaniasis

“…As promastigotes are not sensitive to Sb(V), lines that were reportedly selected for resistance with Sb(V) preparations may have been selected for resistance to the mchlorocresol preservative instead (58,126). Alternatively, Sb(V) preparations could be partially reduced due to prolonged storage at acidic pH or in culture media containing thiols such as cysteine or glutathione (63,65). It is also not inconceivable that some Leishmania spp.…”

“…Both glutathione and trypanothione can nonenzymatically reduce Sb(V) to Sb(III), particularly under acidic conditions (63,65,115,162,163). However, the physiological relevance of these observations is open to question since the rates of reduction are rather slow.…”

Antimonial-induced Increase in Intracellular Ca2+ through Non-selective Cation Channels in the Host and the Parasite Is Responsible for Apoptosis of Intracellular Leishmania donovani Amastigotes

“…We chose PAT, a trivalent antimonial, because pentavalent antimony is converted to trivalent antimony inside macrophages before it can be cytotoxic to the parasites; therefore, trivalent antimony is the active form of pentavalent antimony (27,28). Prior to assessing the anti-leishmanicidal activities of PAT toward L. donovani amastigotes residing within J774A.1 cells, toxicity of the drug (10, 15, 20, 25, and 50 g/ml) on uninfected J774A.1 cells was evaluated to rule out doses that would affect the host cell.…”

Mechanisms underlying the toxic effects of antimony species in human embryonic kidney cells (HEK-293) and their comparison with arsenic species

“…All suggested mechanisms for these drugs propose the uptake of the organic Sb(V) compound with its subsequent reduction to inorganic Sb(III) within the cell by interacting with free sulfhydryl groups within proteins, such as glutathione (GSH) and Cys (Haldar et al, 2011) (Frezard et al, 2001). It also increases ROS production in a human macrophage cell line (Hansen et al, 2011), causing parasite death.…”